Anti-cocaine catalytic antibody

ABSTRACT

This invention provides a polypeptide comprising a light chain domain which comprises a complementarity determining region 1 having the amino acid sequence RSSXGTITXXNYAN (Seq ID No: 73), a complementarity determining region 2 having the amino acid sequence XNNYRPP (Seq ID No: 74) and a complementarity determining region 3 having the amino acid sequence ALWYSNHWV (Seq ID No: 75), interposed between appropriate framework regions, and linked to said light chain domain a heavy chain domain which comprises a complementarity determining region 1 having the amino acid sequence DYNMY (Seq ID No: 76), a complementarity determining region 2 having the amino acid sequence YIDPXNGXIFYNQKFXG (Seq ID No: 77) and a complementarity determining region 3 having the amino acid sequence GGGLFAX (Seq ID No: 78) interposed between appropriate framework regions, said polypeptide having a conformation suitable for degrading cocaine.

This application is a §371 of PCT International Application No.PCT/US97/10965, filed Jun. 25, 1997, which claims priority of and is acontinuation-in-part of U.S. Ser. No. 08/672,345, filed Jun. 25, 1996,the contents of which are hereby incorporated by reference in theirentirety into the present application.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

Catalytic antibodies have unique potential for the treatment of cocaineaddiction and overdose. Cocaine reinforces self-administration byinhibiting a dopamine re-uptake transporter (1) in the mesolimbocortical“reward pathway”. No antagonist to cocaine is known (2), perhapsreflecting the difficulties inherent in blocking a blocker. As analternative to receptor-based therapeutics, a circulating agent couldinterrupt the delivery of cocaine to its binding site in the brain (3).An agent such as an antibody that merely bound the drug could bedepleted stoichiometrically by complex formation but an enzyme thatbound drug, transformed it and released product would be available foradditional binding. Catalytic antibodies, a novel class of artificialenzyme, are inducible for a wide array of reactions and their substratespecificity is programmable to small molecules such as cocaine (4).

Cocaine detoxification is particularly well suited for a catalyticantibody approach. First, hydrolysis of the benzoyl ester of cocaineyields the biologically inactive products (5) ecgonine methyl ester andbenzoic acid (FIG. 1). The plasma enzyme butyrylcholinesterasedeactivates cocaine in humans (6) by means of this reaction. Second,acyl hydrolysis is the best studied of all antibody-catalyzedtransformations (7,8). Esterase activity approaching that of naturalenzymes has been reported (7) for catalytic antibodies and the largehydrophobic surface of the benzoyl ester is particularly well suited toelicit antibodies with strong binding and catalysis.

It has previously described (9) the first catalytic antibodies todegrade cocaine, Mab 3B9 and Mab 6A12. The antibodies were elicited byan immunogenic conjugate (TSA 1) of a phosphonate monoestertransition-state analog. The rate acceleration of these first artificialcocaine esterases (10²-10³) corresponded in magnitude to their relativestabilization of the ground-state to the transition-state(˜K_(m)/K_(i)). Catalytic antibodies with more potent catalyticmechanisms and with higher turnover rates are possible and, it has beenestimated, necessary for clinical applications. Increased activity canbe pursued either through repeated hybridoma generation or throughmutagenesis of catalytic antibodies in hand. However, sequencing of thevariable domains of Mab's 3B9 and 6A12 revealed 93% homology at thecomplementarity determining regions (see below). Such a lack ofdiversity has been noted previously for catalytic antibodies (10) andlimits the opportunities for improving activity since a particular classof homologous catalytic antibodies may fail to optimize to the desiredactivity. A potential solution to this problem, that would notcompromise the core structure of the analog, would be to vary thesurfaces of the analog rendered inaccessible by attachment to carrierprotein and thereby present distinct epitopes for immunorecognition.

The syntheses of three analogs of cocaine hydrolysis with identicalphosphonate replacements but differing constructions for theimmunoconjugates is now reported. The kinetics and the structuraldiversity of the catalytic antibodies elicited by these analogs has beencharacterized. The preferred catalytic antibodies for mutagenesisstudies have been identified.

SUMMARY OF THE INVENTION

The following standard abbreviations are used throughout thespecification to indicate specific amino acids:

E represents Glutamic acid

S represents Serine

R represents Arginine

G represents Glycine

T represents Threonine

I represents Isoleucine

N represents Asparagine

Y represents Tyrosine

C represents Cysteine

P represents Proline

L represents Leucine

W represents Tryptophan

H represents Histidine

D represents Aspartic Acid

F represents Phenylalanine

Q represents Glutamine

V represents Valine

K represents Lysine

M represents Methionine

A represents Alanine

X represents any amino acid

The invention provides catalytic antibody capable of degrading cocainecharacterized by comprising a light chain wherein the amino acidsequence of complementarity determining region 1 is RSSXGTITXXNYAN (SeqID No: 73), the amino acid sequence of complementarity determiningregion 2 is XNNYRPP (Seq ID No: 74) and the amino acid sequence ofcomplementarity determining region 3 is ALWYSNHWV (Seq ID No: 75) and aheavy chain wherein the amino acid sequence of complementaritydetermining region 1 is DYNMY (Seq ID No: 76), the amino acid sequenceof complementarity determining region 2 is YIDPXNGXXFYNQKFXG (Seq ID No.77) and the amino acid sequence of complementarity determining region 3is GGGLFAX (Seq ID No: 78), wherein X can be any amino acid.

The present invention also provides a catalytic antibody capable ofdegrading cocaine comprising a light chain wherein the amino acidsequence of complementarity determining region 1 is RSSSGTITANNYGS (SeqID No. 40), the amino acid sequence of complementarity determiningregion 2 is VSNNRGP (Seq ID No: 41) and the amino acid sequence ofcomplementarity determining region 3 is ALWNSNHFV (Seq ID No: 42) and aheavy chain wherein the amino acid sequence of complementaritydetermining region 1 is TYYIY (Seq ID No: 67), the amino acid sequenceof complementarity determining region 2 is GMNPGNGVTYFNEKFKN (Seq ID No:68) and the amino acid sequence of complementarity determining region 3is VGNLFAY (Seq ID No: 69).

The present invention also provides a catalytic antibody capable ofdegrading cocaine comprising a light chain wherein the amino acidsequence of complementarity determining region 1 is RSSXSLLYXDGKTYLN(Seq ID No: 79), the amino acid sequence of complementarity determiningregion 2 is LMSTRXS (Seq ID No: 80) and the amino acid sequence ofcomplementarity determining region 3 is QXFXXYPFT (Seq ID No: 81) and aheavy chain wherein the amino acid sequence of complementaritydetermining region 1 is SDYAWX (Seq ID No: 82), the amino acid sequenceof complementarity determining region 2 is YIRXXXXTRYNPSLXS (Seq ID No:83) and the amino acid sequence of complementarity determining region 3is XHYYGXXX (Seq ID No: 84).

The present invention provides a catalytic antibody capable of degradingcocaine comprising a light chain wherein the amino acid sequence ofcomplementarity determining region 1 is KSSQSLLYSDGKTYLN (Seq ID: 43),the amino acid sequence of complementarity determining region 2 isLVSKLDS (Seq. ID: 44) and the amino acid sequence of complementaritydetermining region 3 is VQGYTFPLT (Seq ID: 45) and a heavy chain whereinthe amino acid sequence of complementarity determining region 1 is DHWMH(Seq ID: 70), the amino acid sequence of complementarity determiningregion 2 is TIDLSDTYTGYNQNFKG (Seq ID: 71) and the amino acid sequenceof complementarity determining region 3 is RGFDY (Seq ID: 72).

In another embodiment, the present invention provides a polypeptidecomprising a light chain domain with complementarity determining region1 having amino acid sequence RSSXGTITXXNYAN (Seq ID No: 73),complementarity determining region 2 having amino acid sequence XNNYRPP(Seq ID No: 74) and complementarity determining region 3 having aminoacid sequence ALWYSNHWV (Seq ID No: 75), interposed between appropriateframework regions, said light chain domain being linked to a heavy chaindomain with complementarity determining region 1 having amino acidsequence DYNMY (Seq ID No: 76), complementarity determining region 2having amino acid sequence YIDPXNGXIFYNQKFXG (Seq ID No. 77) andcomplementarity determining region 3 having amino acid sequence GGGLFAX(Seq ID No: 78) interposed between appropriate framework regions suchthat said polypeptide assumes a conformation suitable for degradingcocaine.

In another embodiment, the invention provides a polypeptide comprising alight chain domain with complementarity determining region 1 havingamino acid sequence RSSSGTITANNYGS (Seq ID No. 40), complementaritydetermining region 2 having amino acid sequence VSNNRGP (Seq ID No: 41),complementarity determining region 3 having amino acid sequenceALWNSNHFV (Seq ID No: 42) interposed between appropriate frameworkregions, said light chain domain being linked to heavy chain domain withcomplementarity determining region 1 having amino acid sequence TYYIY(Seq ID No: 67), complementarity determining region 2 having amino acidsequence GMNPGNGVTYFNEKFKN (Seq ID No: 68) and complementaritydetermining region 3 having amino acid sequence VGNLFAY (Seq ID No: 69)interposed between appropriate framework regions such that thepolypeptide assumes a conformation suitable for degrading cocaine.

In another embodiment, the invention provides a polypeptide comprising alight chain domain with complementarity determining region 1 havingamino acid sequence RSSXSLLYXDGKTYLN (Seq ID No: 79), complementaritydetermining region 2 having amino acid sequence LMSTRXS (Seq ID No: 80)and complementarity determining region 3 having amino acid sequenceQXFXXYPFT (Seq ID No: 81) interposed between appropriate frameworkregions, said light chain domain being linked to a heavy chain domainwith complementarity determining region 1 having amino acid sequenceSDYAWX (Seq ID No: 82), complementarity determining region 2 havingamino acid sequence YIRXXXXTRYNPSLXS (Seq ID No: 83) and complementaritydetermining region 3 having amino acid sequence XHYYGXXX (Seq ID No: 84)interposed between appropriate framework regions such that thepolypeptide assumes a conformation suitable for degrading cocaine.

In another embodiment, the invention provides a polypeptide comprising alight chain domain with complementarity determining region 1 havingamino acid sequence KSSQSLLYSDGKTYLN (Seq ID No: 43), complementaritydetermining region 2 having amino acid sequence LVSKLDS (Seq ID No: 44)and complementarity determining region 3 having amino acid sequenceVQGYTFPLT (Seq ID No: 45) interposed between appropriate frameworkregions, said light chain domain being linked to heavy chain domain withcomplementarity determining region 1 having amino acid sequence DHWMH(Seq ID No: 70), complementarity determining region 2 having amino acidsequence TIDLSDTYTGYNQNFKG (Seq ID No: 71) and complementaritydetermining region 3 having amino acid sequence RGFDY (Seq ID No: 72)interposed between appropriate framework regions such that thepolypeptide assumes a conformation suitable for degrading cocaine.

The invention further provides a humanized catalytic antibody.

The invention further provides a humanized catalytic polypeptide.

The invention provides an isolated nucleic acid molecule encoding thelight chain of the antibody. Further, the invention provides an isolatednucleic acid molecule encoding the heavy chain of the antibody.

The invention further provides a nucleic acid molecule encoding a singlechain polypeptide.

The present invention further provides a pharmaceutical composition fordecreasing the concentration of cocaine in a subject which comprises anamount of the claimed antibody effective to degrade cocaine in thesubject's blood and a pharmaceutically acceptable carrier.

The present invention further provides a method of decreasing theconcentration of cocaine in a subject which comprises administering tothe subject an amount of the claimed antibody effective to degradecocaine in the subject's blood.

The present invention further provides a pharmaceutical composition fortreating cocaine overdose in a subject which comprises an amount of theclaimed antibody effective to degrade cocaine in the subject's blood anda pharmaceutical acceptable carrier.

The present invention further provides a method for treating cocaineoverdose in a subject which comprises administering to the subject anamount of the claimed antibody effective to degrade cocaine in asubject's blood and reduce cocaine overdose in the subject.

The present invention further provides a pharmaceutical composition fortreating cocaine addiction in a subject by diminishing an achievableconcentration of cocaine which comprises an amount of the claimedantibody effective to degrade cocaine in the subject and apharmaceutical acceptable carrier.

The present invention further provides a method for treating cocaineaddiction in a subject by diminishing the achievable concentration ofcocaine which comprises administering to the subject an amount of theclaimed antibody effective to degrade cocaine and thereby diminishingthe achievable concentration of cocaine in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Hydrolysis of the benzoyl ester of cocaine. Presumed tetrahydralintermediate formed along the reaction pathway is shown. Generalstructure of a phosphonate monoester analogs of the benzoyl ester: TSA1, TSA 2, TSA 3. TSA 4.

FIG. 2. Synthesis of TSA-1.

FIG. 3. Synthesis of TSA-2.

FIG. 4. Synthesis of TSA-3.

FIG. 5. Plot of log (K_(m)/K_(TSA4)) versus log (k_(cat)/k_(uncat)) forcatalytic antibodies generated by TSA1, 2, and 3. Data represented inthis figure are from Tables 1 and 2. Linear relationship by leastsquares method; r=0.85 excluding Mab 15A10 and 8G4G.

FIG. 6. Alignment of Amino acid sequences of Lambda light chains,wherein

9A(lam9)vari (SEQ ID NO:1) indicates the amino acid sequence of thevariable domain of the Lambda light chain of the antibody 9A3;

19G(lam5) vari (SEQ ID NO:2) indicates the amino acid sequence of thevariable domain of the Lambda light chain of the antibody 19G8;

15A10L Vari (SEQ ID NO:3) indicates amino acid sequence of the variabledomain of the Lambda light chain of the antibody 15A10;

G7(lam4) vari (SEQ ID NO:4) indicates the amino acid sequence of thevariable domain of the Lambda light chain of the antibody 8G4G;

FIG. 7. Alignment of Amino acid sequences of Kappa light chains, wherein

3B9 K vari (SEQ ID NO:5) indicates the amino acid sequence of thevariable domain of the Kappa light chain of the antibody 3B9;

6A12 K vari (SEQ ID NO:6) indicates the amino acid sequence of thevariable domain of the Kappa light chain of the antibody 6A12;

12H(L2)k vari (SEQ ID NO:7) indicates the amino acid sequence of thevariable domain of the Kappa light chain of the antibody 12H1;

2A k vari (SEQ ID NO:8) indicates the amino acid sequence of thevariable domain of the Kappa light chain of the antibody 2A10;

E2(L7) k Vari (SEQ ID NO:9) indicates the amino acid sequence of thevariable domain of the Kappa light chain of the antibody 8G4E.

FIG. 8. Alignment of Amino acid sequence of Heavy chains, wherein

3B9 vari (SEQ ID NO:10) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 3B9;

6A12 heavy (SEQ ID NO:11) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 6A12;

12H H vari (SEQ ID NO:12) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 12H1;

2AH-3 (SEQ ID NO:13) indicates the amino acid sequence of the variabledomain of the heavy chain of the antibody 2A10;

9(H-3)vari (SEQ ID NO:14) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 9A3;

19h6-3 vari (SEQ ID NO:15) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 19G8;

15A10 Vari (SEQ ID NO:16) indicates amino acid sequence of the variabledomain of the heavy chain of the antibody 15A10;

E2(H8) Vari (SEQ ID NO:17) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 8G4E.

G7(H8) vari (SEQ ID NO:18) indicates the amino acid sequence of thevariable domain of the heavy chain of the antibody 8G4G;

FIG. 9. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 15A10 (SEQ ID NO:120,121).

FIG. 10. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 15A10 (SEQ ID NO:2).

FIG. 11. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 19G8 (SEQ ID NO:85). The amino acid sequence is setforth in (SEQ ID NO:86).

FIG. 12. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 19G8 (SEQ ID NO:89). The amino acid sequence is setforth in (SEQ ID NO:90).

FIG. 13. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 9A3 (SEQ ID NO:91). The amino acid sequence is setforth in (SEQ ID NO:92).

FIG. 14. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 9A3 (SEQ ID NO:93). The amino acid sequence is setforth in (SEQ ID NO:94).

FIG. 15. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 8G4G (SEQ ID NO:95). The amino acid sequence is setforth in (SEQ ID NO:96).

FIG. 16. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 8G4G (SEQ ID NO:97). The amino acid sequence is setforth in (SEQ ID NO:98).

FIG. 17. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 3B9 (SEQ ID NO:99). The amino acid sequence is setforth in (SEQ ID NO:100).

FIG. 18. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 3B9 (SEQ ID NO:101). The amino acid sequence is setforth in (SEQ ID NO:102).

FIG. 19. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 6A12 (SEQ ID NO:103). The amino acid sequence is setforth in (SEQ ID NO:104).

FIG. 20. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 6A12 (SEQ ID NO:105). The amino acid sequence is setforth in (SEQ ID NO:106).

FIG. 21. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 2A10 (SEQ ID NO:107). The amino acid sequence is setforth in (SEQ ID NO:108).

FIG. 22. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 2A10 (SEQ ID NO:109). The amino acid sequence is setforth in (SEQ ID NO:110).

FIG. 23. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 12H1 (SEQ ID NO:15).

FIG. 24. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 12H1 (SEQ ID NO:111). The amino acid sequence is setforth in (SEQ ID NO:112).

FIG. 25. Nucleotide sequence of the light chain of the anti-cocainecatalytic antibody 8G4E (SEQ ID NO:115). The amino acid sequence is setforth in (SEQ ID NO:116).

FIG. 26. Nucleotide sequence of the heavy chain of the anti-cocainecatalytic antibody 8G4E (SEQ ID NO:117). The amino acid sequence is setforth in (SEQ ID NO:118).

FIG. 27. The scFv of 3B9 catalytic monoclonal antibody (SEQ ID NO:119).

H1 indicates the complementarity determining region 1 of the heavy chainof the antibody 3B9;

H2 indicates the complementarity determining region 2 of the heavy chainof the antibody 3B9;

H3 indicates the complementarity determining region 3 of the heavy chainof the antibody 3B9;

L1 indicates the complementarity determining region 1 of the light chainof the antibody 3B9;

L2 indicates the complementarity determining region 2 of the light chainof the antibody 3B9;

L3 indicates the complementarity determining region 3 of the light chainof the antibody 3B9;

FLAG indicates an epitope recognized by a known antibody; 6×His iscapable of binding to the metal Nickle; both of the Flag and 6×His areuseful for purifying the scFv.

FIGS. 28A and 28B.

(A) Hydrolysis of cocaine at the benzoyl ester and at the methyl ester.

(B) Presumed tetrahedral intermediate of benzoyl ester hydrolysis andcorresponding phosphonate monoester analog.

FIG. 29. Log dose-response relationship for Mab 15A10 on survival afterLD₉₀ cocaine. Male rats received intravenous saline (n=8), or Mab 15A10at 5 mg/kg (n=5), 15 mg/kg (n=5) or 50 mg/kg (n=5) in total volume 5 mlover 5 min. After 5 min, all animals received an intravenouscatecholamine infusion as described¹⁸ and an infusion of cocaine (16mg/kg) at a rate of 1 mg/kg/min. “Survivors” completed the infusionwithout cardiopulmonary arrest and were observed for one hour afterinfusion. The effect of Mab 15A10 on survival was significant byX-square test (p<0.001).

FIGS. 30A-30D.

Saturation of Mab 15A10 with cocaine.

(A and B) Mean cocaine dose at seizure (A) and at death (B).

(C and D) Plasma concentration of ecgonine methyl ester (EME) (C) andcocaine at death (D). To rats prepared as in FIG. 2, saline (n=17) orMab 15A10 100 mg/kg (n=4) or Mab 1C1 100 mg/kg (n=4) in a total volumeof 5 ml was administered intravenously over 5 min. Cocaine was infusedintravenously at a rate of 1 mg/kg/min until cardiopulmonary arrest.Arterial plasma samples were obtained at death for determination ofecgonine methyl ester and cocaine concentrations. The significance ofdifferences between groups, as described in the text, was determined byWilcoxon's Rank Sign test with Bonferroni's correction for multiplecomparisons.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides catalytic antibody capable of degrading cocainecharacterized by comprising a light chain wherein the amino acidsequence of complementarity determining region 1 is RSSXGTITXXNYAN (SeqID No: 73), the amino acid sequence of complementarity determiningregion 2 is XNNYRPP (Seq ID No: 74) and the amino acid sequence ofcomplementarity determining region 3 is ALWYSNHWV (Seq ID No: 75) and aheavy chain wherein the amino acid sequence of complementaritydetermining region 1 is DYNMY (Seq ID No: 76), the amino acid sequenceof complementarity determining region 2 is YIDPXNGXXFYNQKFXG (Seq ID No.77) and the amino acid sequence of complementarity determining region 3is GGGLFAX (Seq ID No: 78).

The present invention also provides a catalytic antibody capable ofdegrading cocaine comprising a light chain wherein the amino acidsequence of complementarity determining region 1 is RSSSGTITANNYGS (SeqID No. 40), the amino acid sequence of complementarity determiningregion 2 is VSNNRGP (Seq ID No: 41) and the amino acid sequence ofcomplementarity determining region 3 is ALWNSNHFV (Seq ID No: 42) and aheavy chain wherein the amino acid sequence of complementaritydetermining region 1 is TYYIY (Seq ID No: 67), the amino acid sequenceof complementarity determining region 2 is GMNPGNGVTYFNEKFKN (Seq ID No:68) and the amino acid sequence of complementarity determining region 3is VGNLFAY (Seq ID No: 69).

The present invention also provides a catalytic antibody capable ofdegrading cocaine comprising a light chain wherein the amino acidsequence of complementarity determining region 1 is RSSXSLLYXDGKTYLN(Seq ID No: 79), the amino acid sequence of Complementarity determiningregion 2 is LMSTRXS (Seq ID No: 80) and the amino acid sequence ofComplementarity determining region 3 is QXFXXYPFT (Seq ID No: 81) and aheavy chain wherein the amino acid sequence of complementaritydetermining region 1 is SDYAWX (Seq ID No: 82), the amino acid sequenceof complementarity determining region 2 is YIRXXXXTRYNPSLXS (Seq ID No:83) and the amino acid sequence of complementarity determining region 3is XHYYGXXX (Seq ID No: 84).

The present invention provides a catalytic antibody capable of degradingcocaine comprising a light chain wherein the amino acid sequence ofcomplementarity determining region 1 is KSSQSLLYSDGKTYLN (Seq ID No:43), the amino acid sequence of complementarity determining region 2 isLVSKLDS (Seq ID No: 44) and the amino acid sequence of Complementaritydetermining region 3 is VQGYTFPLT (Seq ID No: 45) and a heavy chainwherein the amino acid sequence of complementarity determining region 1is DHWMH (Seq ID No: 70), the amino acid sequence of complementaritydetermining region 2 is TIDLSDTYTGYNQNFKG (Seq ID No: 71) and the aminoacid sequence of complementarity determining region 3 is RGFDY (Seq IDNo: 72).

There are five classes of human antibodies. Each has the same basicstructure consisting of two identical polypeptides called heavy chains(molecular weight approximately 50,000 Daltons and two identical lightchains, (molecular weight approximately 25,000 Daltons).

Each of the five antibody classes has a similar set of light chains anda distinct set of heavy chains.

A light chain is composed of one variable and one constant domain, whilea heavy chain is composed of one variable and three or more constantdomains. The combined variable domains of a paired light and heavy chainare known as the Fv region. The Fv determines the specificity of theimmunoglobulin, the constant regions have other functions. Amino acidsequence data indicate that each variable domain comprises threehypervariable regions or loops, called complementarity determiningregions flanked by four relatively conserved framework regions (24). Thehypervariable regions have been assumed to be responsible for thebinding specificity of individual antibodies and to account for thediversity of binding of antibodies as a protein class.

In another embodiment, the present invention provides a polypeptidecomprising a light chain domain with complementarity determining region1 having amino acid sequence RSSXGTITXXNYAN (Seq ID No: 73),complementarity determining region 2 having amino acid sequence XNNYRPP(Seq ID No: 74) and complementarity determining region 3 having aminoacid sequence ALWYSNHWV (Seq ID No: 75), interposed between approprioateframework regions, said light chain domain being linked to a heavy chaindomain with complementarity determining region 1 having amino acidsequence DYNMY (Seq ID No: 76), complementarity determining region 2having amino acid sequence YIDPXNGXIFYNQKFXG (Seq ID No. 77) andcomplementarity determining region 3 having amino acid sequence GGGLFAX(Seq ID No: 78) interposed between appropriate framework regions suchthat said polypeptide assumes a conformation suitable for degradingcocaine.

In another embodiment, the invention provides a polypeptide comprising alight chain domain with complementarity determining region 1 havingamino acid sequence RSSSGTITANNYGS (Seq ID No. 40), complementaritydetermining region 2 having amino acid sequence VSNNRGP (Seq ID No: 41),complementarity determining region 3 having amino acid sequenceALWNSNHFV (Seq ID No: 42) interposed between appropriate frameworkregions, said light chain domain being linked to heavy chain domain withcomplementarity determining region 1 having amino acid sequence TYYIY(Seq ID No: 67), complementarity determining region 2 having amino acidsequence GMNPGNGVTYFNEKFKN (Seq ID No: 68) and complementaritydetermining region 3 having amino acid sequence VGNLFAY (Seq ID No: 69)interposed between appropriate framework regions such that thepolypeptide assumes a conformation suitable for degrading cocaine.

In another embodiment, the invention provides a polypeptide comprising alight chain domain with complementarity determining region 1 havingamino acid sequence RSSXSLLYXDGKTYLN (Seq ID No: 79), complementaritydetermining region 2 having amino acid sequence LMSTRXS (Seq ID No: 80)and complementarity determining region 3 having amino acid sequenceQXFXXYPFT (Seq ID No: 81) interposed between appropriate frameworkregions, said light chain domain being linked to a heavy chain domainwith complementarity determining region 1 having amino acid sequenceSDYAWX (Seq ID No: 82), complementarity determining region 2 havingamino acid sequence YIRXXXXTRYNPSLXS (Seq ID No: 83) and complementaritydetermining region 3 having amino acid sequence XHYYGXXX (Seq ID No: 84)interposed between appropriate framework regions such that thepolypeptide assumes a conformation suitable for degrading cocaine.

In another embodiment, the invention provides a polypeptide comprising alight chain domain with complementarity determining region 1 havingamino acid sequence KSSQSLLYSDGKTYLN (Seq ID No: 43), complementaritydetermining region 2 having amino acid sequence LVSKLDS (Seq ID No: 44)and complementarity determining region 3 having amino acid sequenceVQGYTFPLT (Seq ID No: 45) interposed between appropriate frameworkregions, said light chain domain being linked to heavy chain domain withcomplementarity determining region 1 having amino acid sequence DHWMH(Seq ID No: 70), complementarity determining region 2 having amino acidsequence TIDLSDTYTGYNQNFKG (Seq ID No: 71) and complementaritydetermining region 3 having amino acid sequence RGFDY (Seq ID No: 72)interposed between appropriate framework regions such that thepolypeptide assumes a conformation suitable for degrading cocaine.

The complementarity determining region of the variable domain of each ofthe heavy and light chains of native immunoglobulin molecules areresponsible for antigen recognition and binding.

It has also been discovered that biosynthetic domains mimicking thestructure of the two chains of an immunoglobulin binding site may beconnected by a polypeptide linker while closely approaching, retainingand often improving their collective binding properties.

The binding site of the polypeptide comprises two domains, one domaincomprises variable domain of an immunoglobulin light chain and the otherdomain comprises variable domain of an immunoglobulin heavy chain. Thetwo domains are linked by a polypeptide. Polypeptides held the twodomains in proper conformation to degrade cocaine.

In a preferred embodiment, the invention provides a hybrid singlepolypeptide chain comprising variable fragment of a light chain and avariable fragment of an heavy chain, wherein the complementaritydetermining regions and the framework regions come from separateimmunoglobulins.

In another preferred embodiment, the present invention a humanizedsingle chain polypeptide the framework regions are of human or mammalianorigin.

The use of mouse non-human antibodies have certain drawbacksparticularly in repeated therapeutic regimens. Mouse antibodies, forexample, do no fix human complement well, and lack other importantimmunoglobulin functional characteristics when used in humans. Perhaps,more importantly, antibodies contains stretches of amino acid sequencesthat will be immunogenic when injected into human patient. Studies haveshown that, after injection of a foreign antibody, the immune responseelicited by a patient against an antibody can be quite strong,essentially eliminating the antibody's therapeutic utility after aninitial treatment.

The present invention thus provides hybrid antibodies such as the“humanized” antibodies (e.g. mouse variable regions joined to human orto other mammalian constant regions) by using recombinant DNAtechnology, capable of degrading cocaine. The claimed hybrid antibodieshave one or more complementarity determining regions from one mammaliansource, and framework regions from human or other mammalian source.

The hybrid antibodies of the present invention may be produced readilyby a variety of recombinant DNA techniques, with ultimate expression intransfected cells, preferably immortalized eukaryotic cells, such asmyeloma or hybridoma cells. Polynucleotides comprising a first sequencecoding for human-like antibody framework regions and a second sequenceset coding for the desired antibody complementarity determining regionscan be produced synthetically or by combining appropriate DNA andgenomic DNA segments.

In order to improve the immunogenicity of the hybrid antibody of thepresent invention, the human-like immunoglobulin, called acceptor, isselected to have one of the most homologous sequences to thecorresponding parts of the immunoglobulin donor. The human-likeimmunoglobulin framework sequence will typically have about 65% to 70%homology or more to the donor immunoglobulin framework sequences.

The hybrid antibodies will typically comprise at least about 3 aminoacids from the donor immunoglobulin addition to the complementaritydetermining regions. Usually, at least one of the amino acid immediatelyadjacent to the complementarity determining regions is replaced. Also,the amino acid in the human framework region of an acceptorimmunoglobulin is rare for that position and the corresponding aminoacid in the donor immunoglobulin is common for that position in humanimmunoglobulin sequences.

Finally, the amino acid which is predicted to be within about 3 Angstromof the complementarity determining region in a three-dimensionalimmunoglobulin model and capable of interacting with the antigen or withthe complementarity determining region of the humanized antibody.

When combined into an hybrid antibody, the humanized light and heavychains or complementarity determining regions and framework regions, ofthe present invention will be substantially non-immunogenic in humansand retain the capacity of degrading cocaine as the donor antibody.

The present invention further provides a pharmaceutical composition fordecreasing the concentration of cocaine in a subject which comprises anamount of the claimed antibody effective to degrade cocaine in thesubject's blood and a pharmaceutically acceptable carrier.

The present invention further provides a method of decreasing theconcentration of cocaine in a subject which comprises administering tothe subject an amount of the claimed antibody effective to degradecocaine in the subject's blood.

The present invention further provides a pharmaceutical composition fortreating cocaine overdose in a subject which comprises an amount of theclaimed antibody effective to degrade cocaine in the subject's blood anda pharmaceutical acceptable carrier.

The present invention further provides a method for treating cocaineoverdose in a subject which comprises administering to the subject anamount of the claimed antibody effective to degrade cocaine in asubject's blood and reduce cocaine overdose in the subject.

The present invention further provides a pharmaceutical composition fortreating cocaine addiction in a subject by diminishing an achievableconcentration of cocaine which comprises an amount of the claimedantibody effective to degrade cocaine in the subject's blood and apharmaceutical acceptable carrier.

The present invention further provides a method for treating cocaineaddiction in a subject by diminishing the achievable concentration ofcocaine which comprises administering to the subject an amount of theclaimed antibody effective to degrade cocaine and thereby diminishingthe achievable concentration of cocaine in the subject's blood.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS FIRST SERIES OF EXPERIMENTS General Methods

Unless otherwise noted, reactions were carried out in oven-driedglassware under an atmosphere of argon. Reagent and solvent transferswere made with oven-dried syringes and needles. Dichloromethane,tetrahydrofuran (THF), and benzene were continuously distilled fromcalcium hydride; a fumehood was used for procedures requiring benzene orchloroform. ³H-phenyl-cocaine was prepared as previously reported (8);radiolabeled materials were handled with appropriate caution. Allreagents were purchased from Aldrich Chemical Co. All chromatographysolvents were obtained commercially and used as received. Reactions weremonitored by analytical thin-layer chromatographic methods (TLC) withthe use of E. Merck silica gel 60F glass plates (0.25 mm). Flashchromatography was carried out with the use of E. Merck silica gel-60(230-400 mesh) as described by Still (29). High-pressure liquidchromatography (HPLC) was performed on a system of Waters 590 using aDynamax-C₈ (21.4×250 mm) column and a detector set at 220 nm. Solventsystem was acetonitrile-water (0.1% trifluoroacetic acid).

All carbon NMR spectra were obtained at ambient temperature on either aBruker AMX-500 (500 MHz) spectrometer equipped with a 5 mm broad bandinverse probe, Varian VXR-300 (300 MHz) or a Varian Gemini Varian (50MHz). All proton NMR spectra (400 MHz) were obtained at ambienttemperature on a Bruker AM-400 spectrometer, chemical shifts (δ) arereported in parts per million relative to internal tetramethylsilane(0.00 ppm). FAB high resolution mass spectrometric analysis wereperformed at Michigan State University, Mass Spectrometry Facility. EIMass spectrometric analysis were performed at Columbia University, MassSpectrometry Facility on a JEOL DX303 HF instrument. All results werewithin 5 ppm of calculated values.

Free TSA 4. Ecgonine methyl ester free base was generated by passing aMeOH solution of ecgonine methyl ester hydrochloride through an AmerliteIRN methoxide-exchange column (Polyscience, Inc). To ecgonine methylester (0.049 g, 0.25 mmol) in CH₂Cl₂ (10 ml) at 0° C. were addedphenylphosphonic dichloride (0.042 ml, 0.30 mmol), 1H-tetrazole(catalytic) and N,N-diisopropylethyl amine (0.11 ml, 3.4 mmol). Thereaction was allowed to warn to room temperature. After stirring for 12h, MeOH (0.150 ml) was added and after 4 h the reaction was concentratedin vacuo. Chromatographic purification (SiO₂, CHCl₃/MeOH 99:1) affordedthe mixed diester 4 (0.042 g, 52%) as an oil. To the methyl ester of 4(0.030 g, 0.095 mmol) dissolved in CH₂Cl₂ (3 ml) was addedtrimethylsilyl bromide (0.05 ml, 0.38 mmol) at room temperature for 2 h.The reaction was concentrated in vacuo. Water (5 ml) was added and thereaction was extracted with CHCl₃ (5 ml×2). The organic portions wereextracted with another 5 ml of water. The combined aqueous fractionswere concentrated in vacuo. The residue was taken up in MeOH (5 ml) andpropylene oxide (excess) was added. After concentration in vacuo, thefree TSA 4 (29 mg, 90%) was precipitated as a white solid from asolution of the crude product in CHCl₃. ¹H NMR (400 MHz, D₂O) δ 7.51 (m,2H), 7.32 (m, 3H), 4.37 (m, 1H), 3.83 (m, 1H), 3.67 (m, 1H), 3.54 (s,3H), 2.95 (m, 1H), 2.54 (s, 3H), 2.14-1.92 (m, 3H), 1.91-1.74 (m, 3H).¹³C NMR (300 MHz, D₂O) δ 179.21, 139.31, 136.92, 136.43, 136.30. 134.00,133.81, 69.24, 69.04, 68.57, 58.45, 53.49, 43.96, 40.17, 28.95, 27.83;high resolution mass spectrum (FAB) for C₁₆H₂₃NO₄P (M+1) calcd 340.1314,found 340.1319.

Compound 5. To ecgonine HCl (0.35 g, 1.6 mmol) in MeOH (4 ml) were addedDMF (40 ml), Me₄NOH (2.7 ml, 6.4 mmol), and 1-azido-4-iodobutane (1.8 g,8 mmol). The reaction was stirred at 50° C. for 12 h and thenconcentrated in vacuo. Chromatographic purification (SiO₂,EtOAc/MeOH/NH₄OH 9:0.9:0.1) afforded the ester (0.35 g, 78%) as an oil:¹H NMR (400 MHz, CDCl₃) δ 4.23 (m, 1H), 4.12 (m, 1H), 3.81 (m, 1H), 3.58(m, 1H), 3.26 (t, 2H, J=7.0 Hz), 3.18 (m, 1H), 2.74 (t, 1H, J=4.7 Hz),2.19 (s, 3H), 2.03 (m, 2H), 1.98-1.63 (m, 6H), 1.61-1.47 (m, 2H); ¹³CNMR (500 MHz, CDCl₃) δ 173.73, 64.37, 64.29, 63.56, 61.58, 51.74, 50.94,41.23, 40.26, 25.92, 25.61, 25.51, 24.82; high resolution mass spectrum(FAB) for C₁₃H₂₃N₄O₃ (M+1) calcd 283.1770, found 283.1783.

Compound 6. To alcohol 5 (0.43 g, 1.5 mmol) in benzene (10 ml) at 0° C.,were added phenylphosphonic dichloride (0.27 ml, 1.7 mmol), 1H-tetrazole(8 mg), and N,N-diisopropylethyl amine (0.6 ml, 3.4 mmol). The reactionwas allowed to warm to room temperature and a precipitate was observedafter 15 min. After stirring for 12 h, MeOH (0.1 ml) was added and after4 h the reaction was concentration in vacuo. Chromatographicpurification (SiO₂, CHCl₃/MeOH/NH₄OH 9.5:0.5:0.02), afforded the mixeddiester as a mixture of diastereomers (0.53 g, 89%) as an oil: ¹H NMR(400 MHz, CDCl₃) δ 7.73 (m, 2H), 7.60 (m, 1H), 7.49 (m, 2H), 5.09 (m,1/2H), 4.98 (m, 1/2H), 4.24 (m, 2H), 4.15-3.96 (m, 2H), 3.71 (d, 3/2H,J=14.6 Hz), 3.68 (d, 2H, J=14.6 Hz), 3.35-3.15 (m, 3H), 2.91 (s, 3/2H),2.89 (s, 3/2H), 2.87 (t, 1/2H, J=7.5 Hz), 2.59 (t, 1/2H, J=7.5 Hz),2.43-2.22 (m, 5/2H), 2.17-1.95 (m, 5/2H), 1.71-1.57 (m, 2H), 1.39 (m,2H); ¹³C NMR (500 MHz, CDCl₃) δ 161.55, 149.12, 134.32, 132.55, 129.80,129.66, 66.72, 66.54, 66.45, 66.28, 64.80, 63.90, 63.81, 53.81, 51.60,51.50, 49.58, 49.15, 40.30, 35.60, 35.27, 26.35, 26,06, 26.02, 25.82,25.10, 23.98; high resolution mass spectrum (FAB) for C₂₀H₃₀N₄O₅ (M+1)calcd 437.1954, found 437.1953.

Compound 7. Me₃P (1.1 ml, 1M in THF, 1.1 mmol) was added to azide 6(0.217 g, 0.5 mmol) in 6 ml THF/MeOH/H₂O (9:9:2) and the reaction wasstirred at room temperature for 5 h. After concentration in vacuo, thecrude unstable amine (36 mg, 0.084 mmol) was taken up in dry CH₂Cl₂ (5ml) and 1,4-¹⁴C-succinic anhydride (9 mg, 0.093 mmol) was added. Thereaction was stirred under Ar for 12 h and then concentrated. Forpurification, the crude acid 7 (44 mg, 0.087 mmol) was esterified inCH₂Cl₂ (10 ml) with DCC (36 mg, 0.17 mmol), benzyl alcohol (36 μl, 0.35mmol), and DMAP (cat). The reaction was stirred for 12 h andconcentrated. Chromatographic purification (SiO₂, 0.5:99.5 MeOH/CHCl₃and 2:98 MeOH/CHCl₃) afforded the benzyl ester of 7 as a mixture ofdiastereomers (32 mg, 59%) as an oil. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (m,2H), 7.62 (m, 1H), 7.49 (m, 2H), 7.33 (m, 5H), 6.64 (br, s, 1/2H), 6.56(br. s, 1/2H), 5.10 (s, 2H), 4.96 (m, 1/2H), 4.89 (m, 1/2H), 4.38-3.85(m, 4H), 3.74 (d, 3/2H, J=15.2 Hz), 3.68 (d, 3/2H, J=15.2 Hz), 3.32-3.12(m, 3H), 2.89 (s, 3/2H), 2.87 (s, 3/2H), 2.70-2.59 (m, 3H), 2.52-2.26(m, 4H), 2.10-1.97 (m, 2H), 1.68 (m, 1H), 1.55 (m, 1H), 1.38 (m, 2H);¹³C NMR (500 MHz, CDCl₃) δ 173.55, 172.66, 171.37, 161.62, 161.28,136.59, 134.17, 132.37, 129.56, 129.24, 128.88, 128.71, 67.04, 66.81,66.64, 66.25, 64.66, 63.75, 53.74, 49.37, 49.00, 40.11, 39.42, 35.55,35.26, 31.35, 30.31, 26.19, 26.06, 24.89, 23.91; high resolution massspectrum (FAB) for C₃₁H₄₂N₂O₈P (M+1) calcd 601.2679, found 601.2682.

The benzyl ester of 7 (17 mg, 0.028 mmol) in methanol (10 ml) wasstirred with a catalytic amount of Pd on C (10%) under H₂ (1 atm) for 4h. The reaction mixture was filtered and concentrated in vacuo toprovide acid 7 quantitatively. ¹H NMR (400 MHz, CD₃OD) δ 7.69 (m, 2H),7.60 (m, 1H, 7.51 (m, 2H), 4.99 (m, 1H), 4.20-4.08 (m, 2H), 3.89 (m,1H), 3.73 (d, 3/2H, J=21.5 Hz), 3.66 (d, 3/2H, J=21.5 Hz), 3.62 (m, 1H),3.22 (m, 1H), 3.10 (m, 1H), 3.01 (m, 1H), 2.76 (s, 3/2H), 2.75 (s,3/2H), 2.50 (m, 2H), 2.38-2.28 (m, 5H), 2.04 (m, 2H), 1.61 (m, 1H), 1.50(m, 1H), 1.34 (m, 3H); ¹³C NMR (500 MHz, CD₃OD) δ 176.22, 174.52,173.47, 162,22, 134.97, 132.79, 130.18, 67.66, 67.53, 66.99, 65.47,64.44, 53.89, 39.63, 39.33, 35.99, 31.50, 30.23, 26.71, 24.65, 23.67;high resolution mass spectrum (EI) for C₂₄H₃₆N₂O₈P calcd 511.2209 (M+1),found 511.2218.

Compound 8. To the acid 7 (40 mg, 0.078 mmol) dissolved in acetonitrile(5 ml) was added N-hydroxyphthalimide (14 mg, 0.086 mmol) and DCC (32mg, 0.16 mmol). After 1 h at room temperature a white precipitateformed. The reaction was concentrated in vacuo. The crude activatedester was taken up in CH₂Cl₂ (5 ml) and trimethylsilyl bromide (100 μl,0.78 mmol) was added. The reaction was stirred for 1 h and concentratedin vacuo. The crude reaction mixture was taken up in acetonitrile (5 ml)and amylamine (100 μl, 0.78 mmol) was added. A bright orange colordeveloped immediately and faded to light yellow in 1 h. Another portionof amylamine (100 μl) was added. The reaction was stirred for 12 h atroom temperature and concentrated in vacuo. Water (3 ml) was added andthe reaction was extracted with CHCl₃ (5 ml×2). The organic portionswere extracted with another 5 ml of water. The combined aqueousfractions were concentrated in vacuo. High pressure liquidchromatography on a Dynamax 300 Å, 12μ, C-8 (10×250 mm) column elutingwith 4%-40% CH₃CN/H₂O gradient (0.1% trifluoroacetic acid) provided theamide 8 (16 mg, 36% yield). ¹H NMR (400 MHz, CD₃OD) δ 7.72 (m, 2H), 7.56(m, 1H), 7.47 (m, 2H), 4.12 (m, 3H), 3.87 (m, 1H), 3.23 (m, 2H), 3.14(m, 3H), 2.77 (m, 4H), 2.58 (m, 4H), 2.34 (m, 3H), 2.16 (m, 1H), 1.97(m, 2H), 1.55-1.48 (m, 6H), 1.26 (m, 4H), 0.846 (t, 3H, J=6.3 Hz); ¹³CNMR (500 MHz, CD₃OD) δ 175.76, 173.62, 133.83, 132.23, 131.01, 129.07,66.56, 66.52, 65.26, 64.33, 41.13, 40.36, 39.33, 35.93, 31.13, 29.91,29.48, 28.95, 26.57, 26.28, 24.73, 23.66, 23.22; high resolution massspectrum (FAB) for C₂₈H₄₅N₃O₇P calcd 566.2995 (M+1), found 566.2997.

TSA 1. Acid 7 (14 mg, 0.027 mmol) in CH₃CN (5 ml), was stirred at roomtemperature with N-hydroxyphthalimide (4.8 mg, 0.029 mmol) and DCC (11mg, 0.053 mmol). A red color developed immediately. After 2.5 h, thereaction was partially concentrated in vacuo, filtered through a smallcotton plug and then fully concentrated. The crude, unstable activatedester (0.027 mmol assumed) was taken up in CH₂Cl₂ (5 ml) andtrimethylsilyl bromide (20 μl, 0.15 mmol) was added. The reaction wasstirred for 1 h and concentrated in vacuo. BSA (5 mg) or ovalbumin (5mg) in NaHCO₃ (5 ml, 1 N, pH 8.0) at 0° C. was added and the mixturevigorously stirred. The reaction was allowed to warm to room temperatureand, after 1 h, terminated by gel filtration chromatography (SephadexG-25 M, pH 7.4 PBS). Protein-containing fractions were combined anddialyzed against PBS at 4° C. overnight (pH=7.4, 3×1000 ml). Thecoupling efficiency was estimated to be 6:1 for BSA and 15:1 forovalbumin based on incorporation of radiolabel.

Compound 9a. To 2-(p-bromophenyl)ethanol (1.3 g, 6.5 mmol) were addedmethylene chloride (20 ml), t-butyldimethylsilyl chloride (1.07 g, 7.1mmol) and imidazole (660 mg, 9.7 mmol). The reaction was stirred at roomtemperature for 12 h, filtered and concentrated in vacuo.Chromatographic purification (SiO₂ 95:5 hexane:CHCl₃) afforded the silylether (1.28 g, 66%). To the ether (792 mg, 2.51 mmol) in THF (25 ml)under Ar at −78° C. was added n-BuLi (1.2 ml, 2.3 M hexanes, 2.76 mmol)dropwise. The reaction was stirred for 30 min and a solution ofdiethylchlorophosphate (370 μl, 2.5 M THF, 0.93 mmol) was added. Thereaction was stirred at −78° C. for an additional 5 min and allowed towarm to room temperature. Aqueous NH₄Cl (20 ml) was added and thereaction was extracted with EtOAc (3×10 ml). The combined organic layerswere washed with brine, dried with anhydrous MgSO₄, filtered, andconcentrated in vacuo. THF (10 ml) and aq Bu₄NF (2.5 ml, 1 M, 2.5 mmol)were added to the residue. This solution was stirred at room temperaturefor 30 min and concentrated in vacuo. Chromatographic purification(SiO₂, 9:1 EtOAc/MeOH), provided the alcohol 9a (229 mg, 35%). ¹H NMR(400 MHz, CDCl₃) δ 7.74 (dd, 2H, J=12.5, 7.1 Hz), 7.33 (dd, 2H, J=12.5,4.5 Hz), 4.11 (m, 4H), 2.92 (t, 2H, J=6.5 Hz), 2.89 (t, 2H, J=6.5 Hz),1.32 (t, 6H, J=7.8 Hz). ¹³C NMR (50 MHz, CDCL₃) δ 144.32, 132.51,129.78, 129.47, 63.61, 62.69, 39.74, 16.98; high resolution massspectrum (El) for C₁₂H₂₀O₄P calcd 259.1099 (M+1), found 259.1092.

Compound 9b. To alcohol 9a (193 mg, 0.75 mmol) were added CH₂Cl₂ (7.5ml), Et₃N (115 μl, 0.83 mmol), TsCl (145 mg, 0.75 mmol), DMAP(catalytic). The reaction was stirred at room temperature for 12 h.Concentration and purification (SiO₂, 3:1 EtOAc:hexane) provided thetosylate (251 mg, 81.5%) and to a portion of this product (232 mg, 0.56mmol) were added benzene (3 ml), water (3 ml), tricaprylmethyl ammoniumchloride (cat.), and NaN₃ (150 mg, 2.25 mmol). The reaction was refluxedat 65° C. for 12 h. Saturated aq NH₄Cl (5 ml) was added, and thereaction was extracted with EtOAc. The combined organic layers weretreated with MgSO₄, filtered, and dried in vacuo. Chromatography (SiO₂,1:1 hexane:EtOAc) afforded the azide 9b (137 mg, 86%) ¹H NMR (400 MHz,CDCl₃) δ 7.74 (dd, 2H, J=12.5, 7.1 Hz), 7.32 (dd, 2H, J=12.5, 4.5 Hz),4.09 (m, 4H), 3.86 (t, 2H, J=7.5 Hz), 2.92 (t, 2H, J=7.5 Hz), 1.32 (t,6H, J=7.3 Hz). ¹³C NMR (50 MHz, CDCl₃) δ 143.31, 132.65, 129.50, 129.20,125.31, 62.58, 52.47, 35.89, 16.94; high resolution mass spectrum (EI)for C₁₂H₁₉N₃O₃ P calcd 284.1164 (M+1), found 284.1168.

Compound 10. Diethyl phosphonate ester 8b (600 mg, 2.12 mmol) in CH₂Cl₂(5 ml) were stirred with trimethylsilyl bromide (1 ml, 11 mmol) andwarmed to 45° C. After 20 min, it was concentrated in vacuo. The residuewas dissolved in CH₂Cl₂ (3.2 ml), oxalyl chloride (3.2 ml, 2M in CH₂Cl₂,6.36 mmol) and one drop of DMF were added. After stirring 20 min at roomtemperature, the volatiles was removed in vacuo. The unstable phosphonicdichloride was used directly.

Compound 11. Ecgonine methyl ester free base was generated as describedfor compound 4. To ecgonine methyl ester (170 mg, 0.854 mmol) in benzene(20 ml) at 0° C. was added N,N-diisopropylethylamine (0.74 ml, 4.26mmol), 1H-tetrazole (catalytic) and the phosphonic dichloride 10 (225mg, 0.854 mmol). The reaction was allowed to warm to room temperatureand stirred for 12 h. Methanol (3 ml) was added and after 20 min thereaction mixture was concentrated in vacuo. Chromatographic purification(SiO₂, 1:9 MeOH:CHCl₃) afforded the mixed diester as a mixture ofdiastereomers (108 mg, 30%). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (m, 2H),7.29 (m, 2H), 4.63 (m, 1H), 3.73 (s, 3/2H), 3.70 (s, 3/2H), 3.63 (d,3/2H, J=11.4 Hz), 3.62 (d, 3/2H, J=11.4 Hz), 3.51 (t, 2H, J=7.2 Hz),3.48-3.39 (m, 1H), 3.23-3.15 (m, 1H), 3.05 (m, 1/2H), 2.91 (t, 2H, J=7.2Hz), 2.75 (m, 1/2H), 2.57-2.26 (m, 1H), 2.14 (s, 3H), 2.09-1.52 (m, 5H).¹³C NMR (50 MHz, CDCl₃) δ 170.91, 170.65, 143.27, 132.80, 132.61,129.45, 129.11, 125.08, 78.22, 77.73, 76.95, 70.15, 65.31, 62.14, 52.50,52.84, 52.15, 41.56, 37.84, 35.97, 25.70, 25.58; high resolution massspectrum (EI) for C₁₉H₂₇N₄O₅P calcd 422.1719 (M⁺), found

Compound 12. To azide 11 (370 mg, 0.877 mmol) was added THF (9 ml) andtriphenylphosphine (400 mg, 1.75 mmol). After stirring at r.t. for 12 h,water (1 ml) was added. The mixture was stirred for 3 h and concentratedin vacuo. To the crude amine (200 mg, 0.51 mmol) were added CH₂Cl₂ (7.5ml) and succinic anhydride (3.5 mg, 0.35 mmol). The reaction was stirredfor 12 h and concentrated in vacuo. The crude acid 12 (290 mg, 0.51mmol) was dissolved in CH₂Cl₂ (10 ml) and DCC (200 mg, 0.97 mmol), DMAP(catalytic) and benzyl alcohol (0.2 ml, 1.9 mmol) were added. Thereaction was stirred at room temperature for 12 h and concentrated invacuo. Chromatography SiO₂, 10:10:0.4 CHCl₃:EtOAc:NH₄OH) afforded thebenzyl ester of 12 (197 mg, 65%) as a mixture of diastereomers. ¹H NMR(400 MHz, CDCl₃) δ 7.79-7.61 (m, 4H), 7.33-7.25 (m, 5H), 5.11 (s, 2H),4.69-4.58 (m, 1H), 3.73 (s, 3/2H), 3.69 (d, 3/2H, J=18.1 Hz), 3.62 (d,3/2H, J=18.1 Hz), 3.59 (s, 3/2H), 3.46 (m, 2H), 3.27-3.03 (m, 3H), 2.81(t, 2H, J=7.2 Hz), 2.69 (t, 2H, J=6.8 Hz), 2.42 (t, 2H, J=6.8 Hz), 2.15(s, 3H), 2.08-1.80 (m, 3H), 1.69-1.51 (m, 3H). ¹³C NMR (50 MHz, CDCl₃) δ173.35, 171.42, 132.38, 132.11, 129.99, 129.93, 129.80, 129.67, 129.61,129.56, 129.48, 129.94, 128.66, 128.49, 67.07, 66.16, 66.43, 63.40,53.28, 50.49, 50.18, 50.06, 49.64, 49.36, 49.21, 48.79, 39.58, 36.14,31.14, 30.07, 24.73; high resolution mass spectrum (EI) for C₃₀H₃₉N₂O₈Pcalcd 586.2444 (M⁺), found 586.2428.

Acid 12 was quantitatively regenerated from the benzyl ester asdescribed for acid 7 as a mixture of diastereomers. ¹H NMR (400 MHz,CDCl₃) δ 7.74 (m, 2H), 7.60 (m, 1H), 7.49 (m, 2H), 5.02 (m, 1/2H), 4.92(m, 1/2H), 4.24 (m, 2H), 3.83 (s, 3/2H), 3.74 (d, 3/2H, J=12 Hz), 3.67(d, 3/2H, J=12 Hz), 3.51 (s, 3/2H), 2.79 (m, 1H), 2.75 (s, 3/2H), 2.74(s, 3/2H), 2.45 (m, 1H), 2.35 (m, 6H), 2.02 (m, 2H), 1.20 (m, 4H); ¹³CNMR (300 MHz, CD₃OD) δ 175.92, 174.33, 173.72, 147.06, 132.85, 132.72,130.62, 130.41, 129.56, 129.29, 67.31, 65.28, 64.37, 53.69, 53.43,53.24, 41.25, 39.21, 36.42, 35.83, 35.70, 31.35, 30.58, 30.07, 24.52,23.50; high resolution mass spectrum (EI) for C₂₃H₃₄N₂O₈P calcd 497.2053(M+1), found 497.2064.

Compound 13. To the acid 12 (23 mg, 0.049 mmol) dissolved inacetonitrile (5 ml) was added N-hydroxyphthalimide (9 mg, 0.054 mmol)and DCC (20 mg, 0.097 mmol). Reaction with trimethylsilyl bromide (0.65ml, 0.49 mmol) and amylamine (0.57 ml, 0.47 mmol) proceeded by theprotocols developed for compound 8 to yield amide 13 (8 mg, 30% yield).¹H NMR: (400 MHz, CD₃OD) 7.69 (m, 2H), 7.32 (m, 2H), 4.75 (m, 1H), 4.08(m, 1H), 3.86 (m, 1H), 3.71 (s, 3H), 3.39 (m, 3H), 3.14 (m, 2H), 2.82(m, 5H), 2.42 (s, 3H), 2.38-2.22 (m, 4H), 2.13-2.00 (m, 3H), 1.49 (m,2H), 1.32 (m, 4H), 0.91 (t, 3H, J=1.5 Hz) ¹³C NMR (500 MHz, CD₃OD) δ173.39, 159.53, 159.22, 144.10, 132.23, 130.95, 129.61, 117.04, 64.83,64.62, 64.12, 63.92, 62.53, 40.89, 39.54, 36.83, 36.23, 34.31, 31.21,30.52, 30.14, 29.24, 27.94, 23.95, 21.47; high resolution mass spectrumEI for C₂₇H₄₃N₃O₇P calcd 552.2839 (M+1), found 552.2863.

TSA 2. To acid 12 (70 mg, 0.14 mmol) were added DMF (4 ml), DCC (116 mg,0.57 mmol), and N-hydroxyphthalimide (92 mg, 0.57 mmol) at r.t. Thereaction was stirred for 12 h at 4° C., concentrated in vacuo andfiltered through a small cotton plug rinsing with CHCl₃ (10 ml). To analiquot of this solution (2 ml) was added bromotrimethylsilane (0.1 ml,0.76 mmol). Work-up and coupling proceeded by the protocol developed forTSA 1. The coupling efficiency to BSA was 15 to 1; to ovalbumin 10 to 1.

Compound 14. To N-norcocaine (206 mg, 0.713 mmol) andN,N-diisopropyethylamine (186 μl, 1.07 mmol) in THF (30 ml) was added1-azido-4-iodobutane (160 mg, 0.713 mmol) at r.t. The reaction mixturewas heated to 60° C. for 2 days. Concentration in vacuo andchromatographic purification (SiO₂ 1:9 EtOAc hexane) yielded theecgonine ester 14 (205 mg, 75%) as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ 8.02 (d, 2H, J=6.0 Hz), 7.58 (t, 1H, J=6.1 Hz), 7.41 (t, 2H,J=7.0 Hz), 5.25 (m, 1H), 3.70 (s, 3H), 3.68 (m, 1H), 3.50 (m, 1H), 3.28(t, 2H, J=7.4 Hz), 3.03 (m, 2H), 2.43 (m, 1H), 2.26 (m, 2H), 2.04-2.00(m, 2H), 1.86 (m, 1H), 1.73-1.65 (m, 4H), 1.47 (m, 2H); ¹³C NMR (500MHz, CDCl₃) δ 171.47, 166.96, 133.77, 131.24, 130.59, 129.16, 68.10,63.55, 61.24, 52.89, 52.21, 52.05, 53.13, 36.49, 27.29, 26.95, 26.86,26.34; high resolution mass spectrum (FAB) for C₂₀H₂₇N₄O₄ (M+1) calcd387.2032, found 387.2041.

Compound 15. N-substituted cocaine 14 (205 mg, 0.53 mmol) was hydrolyzedwith aq HCl (10 ml, 0.7 N) at 90° C. for 4 h. The mixture was extractedwith ether, concentrated and dissolved in MeOH (25 ml) saturated withHCl (g). After 2 h at 60°, solvent was removed under vacuum, and theresidue was dissolved in MeOH and passed through an Amberlite IRNmethoxide-exchange column (Polysciences, Inc) (1 ml) to generate thecrude free base. Chromatographic purification (SiO₂ 5:95 MeOH:CHCl₃)afforded alcohol 15 (102 mg, 72%). ¹H NMR (400 MHz, CDCl₃) δ 3.80 (m,1H), 3.69 (s, 3H), 3.03 (m, 1H), 3.66 (m, 2H), 3.24 (t, 2H, J=7.2 Hz),3.18 (m, 1H), 2.75 (t, 1H, J=5.1 Hz), 2.21 (m, 1H), 1.95-1.78 (m, 4H),1.61-1.38 (m, 6H); ¹³C NMR (500 MHz, CDCl₃) δ 169.58, 65.55, 62.89,61.27, 53.10, 52.61, 52.26, 52.18, 41.20, 27.36, 27.08, 27.02, 25.83;high resolution mass spectrum (FAB) for C₁₃H₂₃N₄O₃ (M+1) calcd 283.1770,found 283.1779.

Compound 16. To the ecgonine derivative 15 (102 mg, 0.37 mmol) inbenzene (15 ml) at 0° C. were added 1H-tetrazole (catalytic),N,N-diisopropylethyl amine (0.163 ml, 0.94 mmol) and phenylphosphonicdichloride (0.67 ml, 0.47 mmol). The reaction mixture was allowed towarm to room temperature overnight. Excess MeOH was added and themixture was stirred at room temperature for 3 h. Chromatographicpurification (SiO₂ 5:95 of 4% NH₄OH in MeOH and a 1:1 mixture of hexaneand CHCl₃) and prep-TLC (2.5:97.5 MeOH: CH₂Cl₂) afforded the mixeddiester 16 as a mixture of diastereomers (78 mg, 49%). ¹H NMR (400 MHz,CDCl₃) δ 7.66 (m, 2H), 7.62 (m, 1H), 7.49 (m, 2H), 5.08 (m, 1/2H), 4.97(m, 1/2H), 4.32 (m, 1H), 4.18 (m, 1H), 3.88 (s, 3/2H), 3.75 (d, 3/2H,J=16.4 Hz), 3.71 (d, 3/2H, J=16.4 Hz), 3.49 (s, 3/2H), 3.45-3.25 (m,4H), 2.98 (m, 1H), 2.63-2.22 (m, 4H), 2.19-2.01 (m, 2H), 1.92-1.63 (m,4H); ¹³C NMR (500 MHz, CDCl₃) δ 160.10, 159.72, 133.37, 133.23, 131.61,131.53, 131.46, 130.29, 128.86, 128.76, 128.64, 66.76, 63.74, 63.58,62.55, 62.43, 54.46, 54.17, 52.64, 51.67, 49.11, 48.79, 36.57, 36.28,26.91, 25.58, 25.18, 24.18; high resolution mass spectrum (FAB) forC₂₀H₃₀N₄O₅P (M+1) calcd 437.1954, found 437.1928.

Compound 17. Me₃P (0.156 ml, 1 M, in THF, 0.157 mmol) was added to azide16 (12 mg, 0.026 mmol) in MeOH (5 ml) and the reaction was stirred atroom temperature for 2 h. After concentration in vacuo, the crude aminewas taken up in CH₂Cl₂ (5 ml), succinic anhydride (2.6 mg, 0.026 mmol)was added. The reaction mixture was stirred at room temperatureovernight and concentrated. The crude acid 17 was dissolved in CH₂Cl₂(10 ml) and benzyl alcohol (0.05 ml, 0.048 mmol), DCC (10 mg, 0.048mmol), and DMAP (catalytic) was added. The reaction was stirredovernight at r.t. and concentrated. Column chromatography (SiO₂, 5:95MeOH:CH₂Cl₂) and prep-TLC (5:95 MeOH CH₂Cl₂) afforded the benzyl esteras a mixture of diastereomers (11 mg, 70% from 13). ¹H NMR (400 MHz,CDCl₃) δ 7.76 (m, 2H), 7.63 (m, 1H), 7.51 (m, 2H), 7.32 (m, 5H), 7.01(br s, 1H), 5.09 (s, 2H), 5.03 (m, 1/2H), 4.94 (m, 1/2H), 4.29-4.09 (m,2H), 3.83 (s, 3/2H), 3.77 (d, 3/2H, J=17.1 Hz), 3.69 (d, 3/2H, J=17.1Hz), 3.49 (s, 3/2H), 3.38-3.22 (m, 4H), 3.01 (m, 2H), 2.69-2.33 (m, 8H),2.04-1.60 (m, 6H); ¹³C NMR (500 MHz, CDCl₃) δ 172.94, 172.68, 172.09,135.86, 133.30, 131.64, 128.90, 128.78, 128.65, 128.54, 128.17, 128.82,66.24, 65.81, 62.71, 62.54, 61.16, 61.03, 52.95, 51.49, 47.69, 37.64,35.18, 30.41, 29.39, 25.67, 24.00, 23.54, 21.95; high resolution massspectrum (FAB) for C₃₁H₄₂N₂O₈P (M+1) calcd 601.2679, found 601.2676.

Acid 17 was quantitatively regenerated from the benzyl ester asdescribed for acid 7. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (m, 2H), 7.60 (m,1H), 7.48 (m, 2H), 5.02 (m, 1/2H), 4.92 (m, 1/2H), 4.33-4.09 (m, 2H),3.83 (s, 3/2H), 3.74 (d, 3/2H, J=23 Hz), 3.67 (d, 3/2H, J=23 Hz), 3.51(s, 3/2H), 3.33-3.19 (m, 6H), 2.98 (m, 1H), 2.63 (m, 2H), 2.49 (m, 4H),2.34 (m, 2H), 2.06-1.96 (m, 2H), 1.81-1.76 (m, 2H), 1.57 (m, 2H); ¹³CNMR (300 MHz, CDCl₃) δ 175.23, 173.41, 172.06, 133.21, 131.65, 128.90,128.58, 65.87, 62.75, 60.89, 53.30, 52.98, 51.54, 48.16, 47.75, 37.61,31.02, 30.33, 25.76, 24.15, 23.54, 21.92; high resolution mass spectrum(EI) for C₂₈H₃₆N₂O₈P calcd 511.2209 (M+1), found 511.2213.

Compound 18. To acid 17 (6 mg, 0.012 mmol) dissolved in CH3CN (3 ml) wasadded N-hydroxyphthalimide (2.2 mg, 0.013 mmol) and DCC (5 mg, 0.024mmol). Reaction with trimethylsilyl bromide (0.016 ml, 0.12 mmol) andthe amylamine (0.14 ml, 0.012 mmol) proceeded by the protocols developedfor compound 8 to yield amide 4 (4.4 mg, 65%). ¹H NMR: (400 MHz, CD₃OD)δ 7.81 (m, 2H), 7.56-7.38 (m, 3H), 5.95 (m, 1H), 5.39 (m, 1H), 5.05 (m,1H), 4.79 (s, 3H), 4.29-4.12 (m, 6H), 3.61-3.04 (m, 10H), 2.83-2.34 (m,11H), 0.94 (t, 3H, J=7.2 Hz). ¹³C NMR (300 MHz). δ 175.12, 174.98,174.39, 132.49, 129.36, 129.21, 65.79, 64.72, 62.26, 53.33, 52.52,40.44, 39.01, 36.78, 32.17, 31.91, 30.23, 30.14, 27.39, 24.69, 24.32,23.45, 23.22, 14.36; high resolution mass spectrum (FAB) for C₂₈H₄₅N₃O₇P(M+1) calcd 566.2995, found 566.2997.

TSA 3. To the acid 17 (12 mg, 0.023 mmol) and N-hydroxyphthalimide (16mg, 0.096 mmol) in DMF (2 ml) was added DCC (19 mg, 0.096 mmol). Thereaction was stirred at 4° C. overnight, concentrated in vacuo, andfiltered with CHCl₃ (10 ml) The activated ester was kept as a CHCl₃solution (10 ml) at −20° C. and used without purification.Trimethylsilyl bromide (0.050 ml, 0.379 mmol) was added to a 5 mlaliquot of the activated ester at room temperature. Work-up and couplingproceeded by the protocol developed for TSA 1. The coupling ratio to BSAwas 11:1; to ovalbumin 12:1.

Hybridoma Generation

As previously described (9), BALB/c mice were immunized with theanalog-carriers and the immune response was followed by ELISA.Hybridomas were prepared by standard methods (9,17).

Hybridoma cells (˜2×10⁶) were placed either into a mouse peritoneum thathad been pretreated with pristane or into T-150 flask cell culture. Theharvested ascites or cell culture supernatents were subjected toaffinity chromatography on a preparative protein A HPLC column (Bio-Rad)(purity>90% by SDS-polyacrylamide gel electrophoresis). Samples ofcatalytically active antibodies were purified by anion exchange HPLCwith an analytic DEAE column (TOSOH HASS TSK-gel) using 0.02 M Tris anda linear gradient pH 8.8/0.0 M NaCl to pH 7.0/0.3 M NaCl without loss ofcocaine esterase activity.

Protocol for Binding Studies (CIEIA)

Plates were coated with the TSA (tethered to ovalbumin) that elicitedthe catalytic antibody intended for CIEIA. Free TSA 4 or the TSA-relatedamides 8, 13, or 14, were tested for inhibition of antibody binding tothe eliciting TSA by published protocols (20b).

Protocol for Kinetic Measurements

Catalytic antibody in 50 mM phosphate-buffered saline pH 8.0 (except2A10 and 6A12 at pH 7.0) was incubated with ³H-cocaine typically at fiveconcentrations. At three time intervals, aliquots were acidified withcold HCl (aqueous) to a final pH of 2 and partitioned withhexane-diethyl ether (1:1), and the organic phase was assayed byscintillation counting. Background hydrolysis was determined inotherwise identical reactions without antibody, and observed rates werecorrected. Assays were performed in triplicate with standard error <10%.As a control, the release of benzoic acid was confirmed by HPLC(Perkin-Elmer) using an analytical reverse-phase C₁₈ column (VYDAC) withan acetonitrile-water (0.1% trifluoroacetic acid) gradient and thedetector set at 220 nm.

HPLC analysis of a reaction mixture without antibody showed that themethyl ester of cocaine spontaneously hydrolyzes to benzoyl ecgoninewith a t_(½)=20 hours (pH 7). Thus, benzoyl ecognine is not available asa benzoyl esterase substrate at the early reaction times of the³H-cocaine hydrolysis assay and the release of benzoic acid isattributed solely to cocaine hydrolysis.

Amino Acid Sequencing

Light and heavy chains were separated by SDS-polyacrylamide gelelectrophoresis and then electroblotted to a polyvinylidenedifluoridemembrane (30) for direct NH₂-terminal sequencing by automated Edmandegradation on an Applied Biosystems 470A or 477A sequencer. To obtaininternal sequence, separated bands from 2A10, 19G7, 9A3 and 15A10 werereduced with dithiothreitol, alkylated with iodoacetamide, and cleavedwith trypsin (31) in 1M urea, 0.05 M NH₄HCO₃, pH 8.0. The peptidefragments were extracted from the membrane, separated by HPLC(Hewlett-Packard) on a reverse-phase C4 column (VYDAC) using anacetonitrile-water (0.07% trifluoroacetic acid) gradient and sequenced.

Pcr Cloning of Variable Domains

Mouse hybridoma cell lines producing catalytic antibodies were grown to1×10⁸ cells and total RNA was prepared using a microadaptation of theguanidine thiocyanate/phenol procedure (32) and selection on a oligo(dT) cellulose column.

Degenerate and non-degenerate oligonucleotide PCR primers were designedusing amino acid sequences (2A10,15A10) or the data base of Kabat et al.(24). Restriction endonuclease sites were incorporated into the primersat their 5′ prime end to facilitate cloning. The restriction sitesutilized were Eco RI, Spe I, Xba I, or Xho I. The sense and antisenseoligonucleotide primers for light chain (LC) and heavy chain (HC) ofeach hybridoma line were as follows: For 9A3,19G8,15A10,8G4E and 8G4GLC: 5′-GGAATTCCACIA/TC/GICCIGGIGAA/GACIG-3′ and5′GCTCGAGCC/TTCA/GTGIGTIACITGA/GCA-3′. For 3B9,6A12 and 12H1 LC:5′-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3′ and 5′-GCGCCGTCTAGAATTAACACTCATTCCTGT TGAA-3′. For 2A10 LC:5′-GCTCTAGAGCGAT/CATIGTIATGACICAA/GGAT/CGA-3′ and5′-GGAATTCCA/GTTA/GTGICT/CT/CTCA/GTAT/CTCA/GTC-3′. For3B9,6A12,12H1,9A3,19G8,8G4E and 84G4G HC: 5′-AGGTCCAGCTGCTCGAGTCTGG-3′and 5′-AGGCTTACTAGTACAATCCCTGGGCACAAT-3′. For 2A10 HC:5′-TCCCAGGTCCAACTGCAGCAGCC-3′ and 5′-ATAACCCTTGACCAGGCATCC-3′. For 15A10HC: 5′-CCAGTTCCGAGCTCGTGATGACACAGTCTCC-3′ and5′-AGCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3′.

DNA templates were synthesized using 0.5 μg of hybridoma mRNA andMoloney murine leukemia virus reverse transcriptase. Amplifications werecarried out in a Perkin-Elmer/Cads thermal cycler for 30 cycles ofdenaturation (96° C., 1 min), annealing (50° C., 1 min), and extension(72° C., 3 min). The PCR products were purified by electrophoresis in1.5% agarose gel. Isolated PCR products from each reaction weresubcloned into Bluescript plasmid and analyzed by DNA sequence analysisfor the presence of open reading frame. Nucleotide sequences wereassembled using the IBI MacVector 3.0 program.

EXPERIMENTAL RESULTS

Synthesis of Transition-state Analogs

Phosphonate monoesters, which stably mimic the geometry and chargedistribution of the transition-state for 2nd-order ester hydrolysis byhydroxide, have yielded, in some instances, catalytic antibodies of highactivity (8). However, such analogs are also known to idiosyncraticallyfail to elicit any catalytic antibodies and so the rules for analogconstruction must be defined empirically (11). Strategies to improveanalog efficiency have been devised, including “bait and switch” (11)and substrate attenuation (12), but the cost of such expedients is adivergence between analog and substrate structure which results onaverage in catalytic antibodies with higher values for K_(m). Inhalationof vaporized cocaine yields a peak pulmonary vein concentration (13) of10-30 μM and this is less than the K_(m) of most catalytic antibodieswith esterase activity. At a sub-saturating concentration of cocaine, ahigher K_(m) would result in a lower turnover rate and increase thealready limiting requirement for a high k_(cat). Thus, the constructionof a high fidelity analog that differed from cocaine only by aphosphonate replacement at the acyl group and by the incorporation of atether for construction of an immunogenic conjugate has been chosen.Based on their distances from the locus of reaction and their separationfrom each other, three tether sites were chosen: at the methyl ester foranalog 1, the 4′-position of the phenyl group for analog 2, and thetropane nitrogen for analog 3 (FIG. 1). The “free TSA” corresponded tothe untethered structure 4.

The synthesis of TSA 1 began with the commercially available startingmaterial (−)-ecgonine (FIG. 2). Selective alkylation of the carboxylatesalt of (−)-ecgonine with 4-azido-1-iodo-butane yielded ester 5 in 78%yield. The absence of epimerization at C-2 was confirmed by ¹H-nmrspectroscopy. The base labile and sterically hindered alcohol of alkylecgonine 5 reacted smoothly with phenylphosphonic dichloride using theprocedure for 1H-tetrazole catalysis (14) and addition of methanolprovided the phosphonate diester 6 in 89% yield. The tether waselaborated at the azido moiety by reduction to the unstable amine withP(CH₃)₃ and acylation with 1,4-¹⁴C-succinic anhydride. The hemisuccinatewas purified and characterized as the benzyl ester, obtained in 70%yield from 6, and the acid was quantitatively regenerated by catalytichydrogenolysis. Acid 7 was activated as the N-hydroxyphthalimide esterand selectively deesterified at the phosphonate methyl ester withtrimethylsilyl bromide (15). The unstable monophosphonate product wasimmediately coupled to carrier protein to yield TSA-1. Theanalog:carrier coupling ratio was 6:1 for bovine serum albumin (BSA) and15:1 for ovalbumin based on the incorporation of radiolabel intoprotein. In support of our assignment of structure to the carrier-boundanalog, an aliquot of the monophosphonate was coupled to n-amylamine toyield the expected amide 8.

Synthesis of TSA-2 required a phenylphosphonic dichloride appropriatelysubstituted at the 4′ position for elaboration of a tether (FIG. 3).Silylation of 2-(p-bromophenyl) ethanol followed by transmetallationwith n-butyl lithium, quenching with diethyl chlorophosphate anddesilylation provided alcohol 9a in 23% yield. The tosylate of 9a wasdisplaced by azide and transesterification with trimethylsilyl bromide,followed by reaction with oxalyl chloride (16), provided the requiredphenylphosphonic dichloride 10. Using the tetrazole catalysis methoddescribed above, chloride 10 was coupled with ecgonine methyl ester and,after the addition of methanol, the mixed diester 11 was obtained in 25%yield. The tether was elaborated from the azide by a sequence ofreactions identical to that employed for TSA-1.

For the synthesis of TSA-3, (FIG. 4) N-norcocaine was monoalkylated in75% yield and acid hydrolysis followed by reesterification with acidicmethanol provided alcohol 15 in 72% yield. Tetrazole-catalyzed synthesisof mixed phosphonate diester 16 proceeded in 48% yield and the tetherwas elaborated from the azido moiety as described above.

Generation of Anti-cocaine Catalytic Antibodies

Balb/C mice were immunized with individual analogs conjugated to BSA andhigh titer antisera were elicited by each antigen. Monoclonal antibodieswere prepared by standard protocols (9,17) and hybridomas secretinganalog-specific antibodies as determined by an enzyme-linkedimmunosorbent assay (ELISA) were selected. All IgG anti-analogantibodies were subcloned, propagated in ascites or cell culture flasksand purified by protein A affinity column chromatography. Catalyticantibodies were identified by their capacity to release ³H-benzoic acidfrom ³H-phenyl-cocaine. The radiolabeled benzoic acid was convenientlypartitioned from ³H-cocaine by extraction of the acidified reactionmixture into organic solvent. Hydrolysis of cocaine with commerciallyavailable carboxyl esterase provided a positive control and theproduction of benzoic acid was confirmed by high performance liquidchromatography. A total of nine catalytic antibodies out of 107anti-analog antibodies were identified from 9 fusions with TSA 1yielding 6 out of 50 and TSA 3 yielding 2 out of 49. TSA-2 generatedeight anti-analog antibodies of which one was catalytic. Catalyticantibodies were further purified by DEAE anion exchange chromatographyand they retained activity. All enzymes were inhibited completely by 50μM free TSA 4 (see below) and the Fab portion of each antibody testedretained catalytic activity; the potent inhibitor of serum esterases,eserine (18) at 1 mM, did not inhibit the activity of any catalytic mAband 150 μM free TSA 4 did not inhibit the cocaine esterase activitypresent in serum (results not shown).

Characterization of Catalytic Antibodies

The rate of hydrolysis of ³H-phenyl-cocaine in the presence and absenceof each monoclonal antibody as a function of substrate concentration hasbeen determined. Production of radiolabeled benzoic acid at time pointscorresponding to <5% reaction provided initial rates. A saturationkinetics and obtained a linear Lineweaver-Burk plot for each artificialenzyme has been observed. The first-order rate constants (k_(cat)) andMichaelis constants (K_(m)) of the nine catalytic antibodies ranged from0.011 to 2.3 min⁻¹ and from 150 to 3000 μM, respectively, as shown inTable 1.

TABLE 1 Kinetic parameters for the hydrolysis of ³H- cocaine by Mab's.Mab TSA K_(m) (μM) k_(cat) (min⁻¹) k_(cat)/k_(o) 3B9 1 490 0.11 11006A12 1 1020 0.072 880 2A10 1 3000 0.011 420 9A3 1 270 0.015 140 19G8 1900 0.091 830 15A10 1 220 2.3 23000 12H1 2 150 0.16 1500 8G4G 3 530 0.605500 8G4E 3 1200 0.12 1100

Michaelis constant Km; catalytic rate constant, k_(cat); and spontaneousrate k_(o). Assays were performed at the pH that optimizedk_(cat)/k_(c): in general pH 7.8; for 6A12, pH 7.4; for 2A10, pH 7.0.

The rate acceleration of the most active catalytic antibody, Mab 15A10,was higher and the Michaelis constant lower then those previouslyreported (9) for Mab 3B9; this corresponds to almost two orders ofmagnitude improvement in activity at sub-saturating concentrations ofcocaine. It has also been reported previously that Mab 3B9 displayed arate acceleration commensurate with the ratio of K_(m) to the K_(i) forfree TSA 4. This ratio approximates the affinity of antibody forground-state relative to transition-state and in the case of Mab 3B9suggested that the rate acceleration resulted primarily fromtransition-state stabilization (19). The inhibition constant (K_(i)) offree TSA 4 for Mab 15A10 to be 0.23 μM has been determined; the rateacceleration of this catalytic antibody (k_(cat)/k_(uncat)=2.3×10⁴)significantly exceeded K_(m)/K_(i) (9.6×10²).

The dissociation constant K_(TSA) for all the catalytic antibodies bycompetitive inhibition enzyme immunoassay (20) has been determined(CIEIA) as shown in Table 2.

TABLE 2 Competitive Inhibition Enzyme Immunoassay of catalytic Mab's Mab(TSA) K₄ (μM) K₈ (μM) K₁₃ (μM) K₁₈ (μM) 3B9 (1) 0.01 0.02 3 100 6A12 (1)0.01 0.01 4 90 2A10 (1) 0.5 3 20 150 12H1 (2) 0.001 0.01 2 60 9A3 (1)0.05 0.02 — 0.003 19G8 (1) 0.008 0.001 — 0.001 15A10 (1) 0.009 0.003 —0.0005 8G4G (3) 0.003 0.001 — 0.001 8G4E (3) 0.003 0.0005 — 0.003

Dissociation constants for free TSA 4 and TSA-related amides 8, 13, or18 were determined for each catalytic Mab by CIEIA through competitiveinhibition of Mab binding to the TSA (1, 2 or 3 tethered to ovalbumin)that elicited the Mab.

K_(TSA) determined by CIEIA provides a relative measure of K_(i) andpermits assay at very low concentrations of antibody.

As shown in FIG. 1, a log-log plot of k_(cat)/k_(uncat) vs.K_(m)/K_(TSA) displayed a linear relationship (r=0.85) for 7 of the 9catalytic antibodies; since K_(TSA) is proportional to K_(i), therelationship k_(cat)/k_(uncat){tilde over (=)}K_(m)/K_(i) for Mab 3B9 islikely true for all seven antibodies. Mab 15A10 deviated from this line,as expected since k_(cat)/k_(uncat) exceeded K_(m)/K_(i) as describedabove; Mab 8G4G also apparently deviated as shown. Thus, the rateacceleration for 15A₁₀, and perhaps 8G4G, appears too great to be solelyattributed to transition-state stabilization and the participation ofchemical catalysis, such as acid-base or nucleophilic catalysis, islikely.

Mab 15A10 was not inhibited by the product of cocaine hydrolysis,ecgonine methyl ester, at a concentration of 1 mM. Benzoic acid didinhibit with a K_(i) of 250 μM. However, in humans, benzoic acid plasmalevels are markedly suppressed by a rapid and nearly complete conversionto hippuric acid (21). It was found that 1 mM hippuric acid did notinhibit Mab 15A10. Also, there was no inhibition from 1 mM benzoylecgonine, a prominent metabolite of cocaine in man (22). Inactivation ofMab 15A10 by repetitive turnover was not observed; after 6 hrs, and >200turnovers, the k_(cat) remained >95% of baseline. The presence ofminimal product inhibition by ecgonine methylester was fortuitous;heterologous immunization (23) with TSA 1, 2, and 3 and thecorresponding 1,2-aminoalcohol analogs of cocaine is planned both forits potential to minimize product inhibition and its capacity toincrease the yield of active enzymes.

The rationale for varying the tether sites of TSA to carrier protein(BSA) was to expose unique epitopes and elect catalytic antibodiesspecific to each immunogen. In order to assess binding specificity, thecatalytic antibodies were examined by ELISA with TSA 1, 2, and 3 boundto ovalbumin. Unexpectedly, two groups with broad affinities wereidentified, a “3B9 group” (Mab's 3B9, 6A12, 2A10, 12H1) that bound allthree conjugates and a “9A3 group” (Mab's 9A3, 19G8, 15A10, 8G4G, 8G4E)that bound only TSA-1 and 3.

To estimate the affinities for TSA 1, 2, and 3 within these groupsrelative K_(d)'s of the corresponding amides 8, 13, and 18 by CIEIA hasbeen determined. As shown in Table 2, CIEIA confirmed the ELISA result,identifying the same two broad groups of catalytic antibodies. The 3B9group displayed the rank order of affinities: 8>13>18. The relativeK_(d) for the amide of the TSA that elicited each antibody ranged from0.01 μM for Mab 3B9 and 6A12 to 3 μM for Mab 2A10. Mab 12H1 derived fromTSA 2 showed a greater affinity for the TSA1-related amide 8 (0.01 uM)then for the TSA2-related amide 13 (2 uM). TSA 1 could have elicited Mab12H1 and the affinities of Mab's 3B9, 6A12 and 2A10 for 13 are alsoprobably sufficient for TSA 2 to have elicited them. The very lowaffinities of the 3B9 group for the TSA3-related amide 18 suggest thatTSA 3 could not have elicited this group.

The 9A3 group showed a distinctly different pattern with very highaffinity for TSA1-related amide 8 and TSA3-related amide 18 butvirtually none for TSA2-related amide 13. Apparently, TSA-1 or TSA-3could have elicited every member of this group; TSA-2 could not haveelicited any.

To assess the structural diversity of the catalytic Mab's, pcr-cloningand sequencing the variable regions of the heavy and light chains ofeach antibody were performed. Primers were generally derived frompublished consensus sequences (24). The 600-700 bp pcr fragment fromeach reaction was cloned into pBluescript and independently preparedclones were sequenced in both directions. The deduced primary amino acidstructures contained the N-terminal amino acid sequences derived fromauthentic catalytic antibody samples. Amino acid sequencing alsoprovided primers for pcr-cloning of Mab's 2A10 and 15A10. Thecomplementarity determining regions (CDR's) were aligned for comparison(Table 3), and several discrete families of anti-cocaine catalyticantibodies were identified.

TABLE 3 Deduced amino acid sequences of catalytic antibodies light chainCDR's (Panel A) (SEQ ID NOS:19-45) and heavy chain CDR's (Panel B) (SEQID NOS:46-72). Mab CDR1 CDR2 CDR3 A. 3B9 RSSRSLLYRDGKTYLN LMSTRSSQHFVDYPFT 6A12 RSSKSLLYEDGKTYLN LMSTRAS QHFEDYPFT 2A10 RSSKSLLYEDGKTYLNLMSTRAS QQFVEYPFT 12H1 RSSRSLLYRDGKTYLN LMSTRAS QHFEDYPFT 9A3RSSTGTI-TTSN-YAN INNNRPP ALWYSNHWV 19G8 RSSAGTI-TTSN-YAN VNNNRPPALWYSNHWV 15A10 RSSTGTI-TSDN-YAN VNNYRPP ALWYSNHWV 8G4G RSSSGTI-TANN-YGSVSNNRGP ALWNSNHFV 8G4E KSSQSLLYSDGKTYLN LVSKLDS VQGYTFPLT B. 3B9 SDYAWTYIR-HIYGTRYNPSLIS YHYYGS-AY 6A12 SDYAWY YIR-HIYGTRYNPSLIS YHYYGS-AY 2A10SDYAWN YIR-YSGITRYNPSLKS IHYYG-YGN 12H1 SDYAWT YIR-HIYGTRYNPSLISYHYYGS-AY 9A3 -DYNMY YIDPSNGGIFYNQKFKG -G-GGLFAY 19G8 -DYNMYYIDPHNGGIFYNQKFKG -G-GGLFAY 15A10 -DYNMY YIDPSNGDTFYNQKFQG -G-GGLFAF8G4G T-YYIY GMNPGNGVTYFNEKFKN --VGNLFAY 8G4E -DHWMH TIDLSDTYTGYNQNFKG-R-G--FDY

TSA 1 yielded two structural families, 3B9-6A12-2A10 and 9A3-19G8-15A10.The light chain CDR homology for parings within the 3B9 family averaged96%; within the 9A3 family the average was 93%; whereas between thesefamilies the average was 14%. The heavy chain CDR homology within the3B9 family was high with 3B9 and 6A12 identical and 2A10 67% homologous;within the 9A3 family the average heavy chain CDR homology was 88%; butbetween the 3B9 and 9A3 families the average was 32%. TSA 3 yielded twosingle-membered families 8G4G and 8G4E. The light chain CDR homology for8G4G showed 68% homology to the 9A3 group and ≦20% homology to theothers; 8G4E showed 56% homology with the 3B9 group and ≦20% to allothers. The heavy chain CDR homology between 8G4G and BG4E was 24%; foreach to the 9A3 group 48% and <20% to all others. Mab 12H1, derived fromTSA-2, showed high homology (96%) to the light chain CDR's of the3B9-6A12-2A10 group and was identical to the heavy chain CDR's of 3B9and 6A12.

Example of Synthesis of an Single Chain Fv Fragment

Single chain Fv fragments for catalytic monoclonal antibody 3B9 havebeen prepared via the following construction.

Mab 3B9 DNA of V_(H) and V_(L) were subcloned by PCR using followingprimers V_(H):

5′TATCCATATGGAGGTGCAGCTGCAGGAGTCTGGACCTGAGCTGGTGAA GCC3′

and

5′ATGGGGGTGTCGGCATGCCTGCAGAGAC3′;

and the following primers V_(L),

5′CCCCATGGATATTGTGATGACCCAGGAT3′

and

5′TAACTGCTCGAGGGATGGTGGGAA3′.

DNA of V_(L) was digested by Nco I and Xho I and introduced into pET20b(Novagen). DNA of V_(H) was digested by Nde I and SphI, and introducedinto pUC18 containing a following linker sequence:

(SphI)-CATCCGGAGGCGGTGGCTCGGGCGGTGGCGGCTCGGGTGGCTCTGC-(NcoI).

This plasmid was digested by NdeI and NcoI, and introduced into pET20bcontaining V_(L) DNA. Then, this plasmid was digested by Xho I and afollowing sequence that codes flag sequence was introduced;TCGATTACAAGGACGACGATGACAAGC. The resulting plasmid was transformed intoBL21(DE3) pLysS. Cells were grown in LB medium at 37° C. At an OD₅₅₀ of0.6 IPTG was added to a final concentration of 2 mM, and the cells werefurther grown for 2 hrs. before harvest. The cells were suspended in 20of culture volume of binding buffer (5 mM imidazole/0.5M NaCl/20 mMTris-HCl, pH 7.9)/6M Urea, disrupted by freezing and thawing and removeddebris by centrifugation (10000 g×20 min). Supernatant was applied toHistBind Resin Column (Novagen) and eluted with 6M urea/1Mimidazole/0.5M NaCl/20 mM Tris-HCl pH 7.9.

Elisa analysis of the resulting single chain Fv fragment demonstratedbinding activity. Enzymatic activity was confirmed by the release of the³H benzoic acid from the ³H phenyl-cocaine.

EXPERIMENTAL DISCUSSION

The clinical application of a catalytic antibody against cocaine relieson a kinetic argument since a 100 mg dose of cocaine if antagonizedsolely by antibody binding would require 25 g of antibody (assuming anantibody MW of 150 kD and 2:1 cocaine:antibody stoichiometry). Activeimmunization with cocaine tethered to an immunoconjugate would beunlikely to provide more than a few percent of this requirement (25).Polyclonal gamma globulin can be administered in doses of this magnitudebut clearly only enzymatic turnover reduces the antibody requirement toa practical magnitude and, most importantly, allows for the burden ofrepetitive self-administration—the hallmark of addiction.

The optimization of an anti-cocaine catalytic antibody which greatlyreduces the cost per dose can be approached through improved analogdesign, large scale antibody selection (26) and antibody mutagenesis(27). Mab 15A10 and 8G4G are the preferred candidates for optimizationsince they are the most active catalytic antibodies; they arestructurally distinct (see below); and Mab 15A10, and possibly 8G4G,could already manifest some element of chemical catalysis. The failureof decades of effort to identify classical receptor blockers of cocaine,together with the compelling nature of the cocaine problem, justify anexhaustive strategy employing all three approaches. One impediment tothis effort is the limited diversity of the antibodies elicited by agiven analog. Clearly, antibody diversity is not necessary if, bychance, a single class of antibodies ultimately yields a member with thedesired kinetic parameters. However, the capacity of a given antibody tobe optimized to specification cannot be predicted due to the scarcity ofstructural data on catalytic antibodies. The generation of a diversegroup of anti-cocaine catalytic antibodies should improve the prospectsfor successful optimization whether through repetitive large-scalehydridoma preparation or through mutagenesis.

Using the tetrazole catalysis method for phosphonate ester synthesis,three transition-state analogs of cocaine hydrolysis were synthesized.The core phosphonate monoester structure was identical in each and onlythe tether sites varied. All three elicited catalytic antibodies and acompetitive ELISA and CDR sequencing were used to define functional andstructural groupings, respectively.

A comparison of the CDR's of the active antibodies delineated fourdiscrete non-overlapping families that were elicited specifically by TSA1 (3B9-6A12-2A10 and 9A3-19G8-15A10) and TSA 3 (8G4G and 8G4E). TSA 2yielded one antibody highly homologous to the 3B9-6A12-2A10 family fromTSA 1 and without homology to the antibodies derived from TSA 3. Thesestructural families overlapped in part with two broad groups defined bya CIEIA method in which amides 8, 13, and 18 (representing TSA 1, 2 and3, respectively) inhibited the binding of each catalytic antibody to itseliciting TSA.

One group defined by CIEIA consisted of Mab's 3B9, 6A12, 2A10 and 12H1.This group displayed high affinity for 8, moderate affinity for 13 andvery low affinity for 18. All of the highly homologous members of thisgroup could have been elicited by TSA 1; the one antibody derived fromTSA 2, Mab 12H1, bound TSAl-related amide 8 with even greater affinitythan TSA2-related amide 13. Nonetheless it is possible that most if notall of the group could have been elicited by TSA 2 since the range ofaffinities for 13 in this group overlapped with the range of affinitiesfor the amides of the TSA's that elicited each antibody. In contrast,the very low affinity of 18 for every member of this group suggests thatTSA 3 could not yield any member of the group. A strategy to obtaincatalytic antibodies against cocaine based only on a TSA tethered at thetropane nitrogen (28) would fail to identify this group of antibodies.

The second group defined by CIEIA consisted of five catalytic antibodiesfrom three structural families: 9A3-19G8-15A10 derived from TSA 1; 8G4Gand 8G4E from TSA 3. These five antibodies displayed equally highaffinity for amides 8 and 18 and in principle either TSA 1 or 3 couldhave elicited every catalytic antibody in this group. That TSA 1 and 3did not yield members of a common structural family may reflect theinadequacy of a sample size averaging 3 fusions per analog. None of thefive antibodies could have been obtained with TSA 2 and thus three ofthe four structural families would not have been identified with thisconjugate.

TSA 1 elicited the most active catalytic antibody, Mab 15A10. Moreover,based on the high affinity of amide 8 for all nine catalytic antibodies,TSA 1 could plausibly have elicited every antibody described. Thisresult was unexpected but not a definitive endorsement of TSA 1 as thepreferred analog. With more aggressive screening, TSA 2 or 3 mayultimately yield a more active antibody not recognized by TSA 1.

Clearly, the failure of a TSA (e.g. TSA 2) to bind to a catalyticantibody (e.g. 15A10) derived from an alternate immunogenic conjugateconfirms that the location of the tether limits the catalytic antibodiesproduced and supports varying the site of attachment to carrier protein.Exhaustive screening of hybridomas from TSA 1, 2 and 3 and detailedstructural studies of the catalytic antibodies elicited may clarify therules for analog construction. The pursuit of high activity anti-cocainecatalytic antibodies provides a compelling justification for thiseffort.

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8. Benkovic, S. J.; Adams, J. A.; Borders, C. C. Jr.; Janda, K. D.;Lerner, R. A. Science, 1990, 250, 1135; (b) Tramontano, A.; Ammann, A.A.; Lerner, R. A. J. Am. Chem. Soc. 1988, 110, 2282.

9. Landry, D. W.; Zhao, K.; Yang, G. X.-Q.; Glickman, M.; Georgiadis, T.M. Science, 1993, 259, 1899.

10. Miyashita, H.; Hara, T.; Tanimura, R.; Tanaka, F.; Kikuchi, M.;Fujii, I. Proc. Natl. Acad. Sci. USA. 1994, 91, 6045.

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SECOND SERIES OF EXPERIMENTS Introduction

Cocaine overdose, a potentially fatal syndrome, has long defieddevelopment of antagonists. To provide a new approach, a high activitycatalytic antibody was elicited using a transition-state analog for thehydrolysis of cocaine to non-toxic products. This antibody protectedrats from cocaine-induced seizures and sudden death in a dose-dependentfashion. Consistent with accelerated catalysis, the hydrolysis productecgonine methyl ester was increased >10-fold in plasma; a non-catalyticanti-cocaine antibody did not reduce toxicity. This artificial cocaineesterase is the first rationally designed cocaine antagonist and thefirst catalytic antibody with potential for medicinal use.

Cocaine is presently abused in the United States by approximately twomillion hardcore addicts and over four million regular users (1). Theacute toxicity of cocaine overdose frequently complicates abuse and thepotential medical consequences of this syndrome include convulsions anddeath (2). Despite decades of effort, however, no useful antagonists tococaine have been found. This failure is due, in part, to the drug'sunique mechanism of action as a competitive blocker of neurotransmitterre-uptake (3). Thus, cocaine's blockade of a dopamine re-uptaketransporter in the central nervous system (CNS) is hypothesized to causereinforcement (4) and the difficulties inherent in blocking a blockerappear to have hindered the development of antagonists for addiction.For cocaine overdose this problem is compounded by the binding ofcocaine at high concentrations to multiple receptors in the CNS andcardiovascular systems. For instance, blockade of serotonin-reuptaketransporters contributes to cocaine-induced convulsions (5,6);dopamine-reuptake blockade (6) and dopamine D₁ receptor binding (7)contribute to lethality; and blockade of norepinephrine-reuptaketransporters, as well as blockade of cardiac myocyte Na⁺ channels andother ion transporters, contributes to arrhythmias and sudden death (8).Thus, cocaine overdose may well pose an insurmountable problem for theclassical receptor-antagonist approach.

These difficulties in developing antagonists for cocaine abuse led to anew approach—to intercept cocaine with a circulating agent therebyrendering it unavailable for receptor binding. An antibody is an obviouschoice for a circulating interceptor but, as noted in the original 1974report on anti-heroin antibodies, the stoichiometric binding of the drugeffectively depletes antibody (9). To overcome the limitations ofbinding, catalytic antibodies were developed—a novel class of artificialenzyme (10)—with the capacity to bind and degrade cocaine, releaseproduct and become available for further binding (11). Since degradationof cocaine at its benzoyl ester yields non-toxic products, ecgoninemethyl ester (12) and benzoic acid (13) (FIG. 28A), a phosphonatemonoester transition-state analog for benzoyl ester hydrolysis (TSA-I,FIG. 28B), was synthesized and with it elicited the first catalyticantibodies to degrade cocaine in vitro (11).

The catalytic activity of these antibodies was insufficient todemonstrate a biologic effect but through repetitive hybridomapreparation with the reagent TSA-I, Mab 15A10, an antibody 100-fold morepotent at sub-saturating concentrations of cocaine (14) was generated.This antibody is the most potent artificial cocaine esterase to datewith a Michaelis constant of 220 μM, a turnover rate of 2.3 min⁻¹, and arate acceleration of 2.3×10⁴. The antibody retained >95% of its activityafter >200 turnovers and product inhibition, a frequent impediment touseful antibody catalysis (15), was not observed for the alcohol productecgonine methyl ester at concentrations up to 1 mM. Although Mab 15A10was inhibited in vitro by benzoic acid (Kd˜250 μM), this acid is rapidlycleared from plasma through coupling to glycine (13,16) and the adduct,hippuric acid, was not an inhibitor in vitro at a concentration of 1 mM.Thus, Mab 15A10 possesses several characteristics essential for apractical in vivo catalyst.

Using Mab 15A10, the antibody-catalyzed degradation of cocaine wastested to see if it could block the acute toxicity of cocaine overdosein rat. The toxicity of cocaine can vary significantly among individualsdepending on endogenous catecholamine levels and this likely explainsthe variably increased incidence of sudden death in restrained animals(17) and agitated patients (18). In previous work (19), catecholaminelevels were standardized through intravenous infusion in conscious,unrestrained animals and, for continuously infused cocaine (1mg/kg/min), found that the LD₅₀ was 10 mg/kg and the LD₉₀ was 16 mg/kg.

Using this method (20), animals pretreated with Mab 15A10 (21) showed asignificant (p<0.001) dose-dependent increase in survival to an LD₉₀cocaine infusion (FIG. 29). Four of five animals receiving antibody at15 mg/kg and all of five receiving antibody at 50 mg/kg survived. Incontrast, all eight rats not treated with Mab 15A10 expired before thecocaine infusion was complete. In the animals not treated with Mab15A10, the mean cocaine dose at death was 7.5±0.6 mg/kg, whereas thefive treated with antibody at 5 mg/kg expired at a mean cocaine dose of8.2±1.0 mg/kg and the single non-survivor in the group treated withantibody at 15 mg/kg expired at 15.9 mg/kg of cocaine.

To further quantify the protective effect of the catalytic antibody, the15A10 (100 mg/kg) and control groups were overwhelmed with intravenouscocaine continuously administered at 1 mg/kg/min until all animalsexpired (FIGS. 30A and 30B). The dose of cocaine at seizure averaged9.48 mg/kg for saline controls and 32.5 mg/kg for animals treated withMab 15A10 (p<0.01) (FIG. 30A). The mean lethal dose of cocaine was alsoincreased over 3-fold, from 11.5 mg/kg of cocaine for controls to 37.0mg/kg for the Mab 15A10 group (p<0.01) (FIG. 30B).

Simple binding was an unlikely explanation for the effectiveness of Mab15A10 since stoichiometric binding of cocaine would be expected to shiftthe dose-response to cocaine by <1 mg/kg. However, to exclude thispossibility, the action of a binding antibody, Mab 1C1, was tested at anequal dose. Mab 1C1 was elicited by immunization with TSA-I, but theantibody is not catalytically active since it binds free TSA and cocainewith comparable affinity (22). As expected, Mab 1C1 was ineffective inblocking cocaine-induced convulsions or death (FIGS. 30A and 30B).

To demonstrate in vivo catalysis, the plasma concentrations of cocainehydrolysis products in the 15A10 and control groups were measured bypreviously developed high-pressure liquid chromatography (HPLC) method(23). The 15A10 group showed a >10-fold increase in ecgonine methylester (24) compared to either the saline (p<0.001) or the Mab 1C1(p<0.01) control groups (FIG. 30C). As expected based on its rapidmetabolism (13,16), plasma benzoic acid concentrations were notsignificantly elevated in the 15A10 group (3.85±0.89 μM) compared to thesaline control group (2.36±1.05 μM) Consistent with specific catalysisat the benzoyl ester, the plasma concentration of the methyl esterhydrolysis product, benzoyl ecgonine (FIG. 28A), was not significantlyincreased in the Mab 15A10 group (7.68±1.07 mM) compared to salinecontrol (5.47±1.01 μM).

Plasma cocaine concentrations in 15A10 and control groups were measuredat death by HPLC (23) in order to confirm that Mab 15A10 conferredresistance to cocaine toxicity through a pre-receptor mechanism. Amarked elevation of plasma cocaine would be expected if Mab 15A10 actedat or after the binding of cocaine to its receptors. In contrast, plasmacocaine concentrations at death were not significantly different between15A10 and control groups (FIG. 30D), as expected for a pre-receptoreffect and consistent with protection from toxicity through catalyzeddegradation of cocaine.

The present study provides a proof of the concept for the use ofcirculating catalytic antibodies to block the toxic effects of cocaine.The incidence of cocaine overdose in the United States is approximately80,000 cases per year and cocaine-related deaths exceed 3,000 per year(1). An anti-cocaine catalytic antibody could be a useful therapeuticfor patients manifesting serious complications of overdose such asseizures and arrhythmias. Mouse monoclonal 15A10, the first catalyticantibody with potential for medicinal use, is a suitable candidate formutagenesis to further improve kinetics (25) and protein engineering toenhance human compatibility (26). Assessment of Mab 15A10 and moreactive homologs in an animal model based on antibody post-treatment ofcocaine toxicity would precede human trials.

Since the original report on anti-cocaine catalytic antibodies (3),others have described variations on the concept of intercepting cocainebefore the drug reaches its receptors. For example, intraperitonealadministration of the enzyme butyrylcholinesterase was shown to inhibittoxicity due to intraperitoneal cocaine in mouse (27). Also,non-catalytic anti-cocaine antibodies were shown to diminishcocaine-induced psychomotor effects and reinforcement in rat (28).However, catalytic antibodies are likely to be longer-lived in plasmathan natural enzymes and, in contrast to typical antibodies, notsusceptible to depletion by complex formation with cocaine. Thus,catalytic antibodies have the unique potential to treat both the acuteand chronic aspects of cocaine abuse and, as a result, practicalexperience with acute overdose can provide a foundation for thetreatment of chronic addiction.

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19. B. Mets, S. Jamdar, D. Landry, Life Sci 59, 2021 (1996).

20. Male rats (350-400 g) were fitted with femoral arterial and venouscatheters under pentobarbital anesthesia. After 24 hrs arterial pressurewas transduced and catecholamines [norepinephrine (0.725 μg/min),epinephrine (0.44 μg/min), and dopamine (0.8 μg/min)] were infusedintravenously with co-infusion of cocaine at one mg/kg/min for 16-min.HPLC measurements of catecholamines levels (9) at baseline and at thetime of cardiopulmonary arrest were found not to be significantlydifferent between groups (p>0.05).

21. Hybridoma 15A10 was seeded in a Fibra Cel cell support matrix(Cellagen Plus bioreactor, New Brunswick Scientific Co, New Brunswick,N.J.) continuously perfused with RPMI 1640 (GIBCO) medium. Perfusate wasconcentrated with a prep. scale 10K 6 sq. ft. cartridge (Millipore) andsubjected to Protein G chromatography to yield Mab 15A10 >90% pure bySDS-PAGE chromatography. Catalytic activity was comparable to thatpreviously described¹⁴ and was completely inhibited by free TSA (50 μM)Endotoxin levels were <0.1EU/ml by QCL—1000 quantatitive chromogenic LALassay.

22. Mab 1C1 was obtained from the original hybridoma preparation withTSA-I as described(14). For Mab 1C1, the cocaine IC₅₀ was 30 μM byinhibition of ³H-cocaine binding (31 mCi/mmol, New England Nuclear,Waltham, Mass.) with cold cocaine 0-1000 μM in phosphate buffered saline(pH 7.4). Bound radiolabel was separated from free by gel filtrationchromatography using standard methods: D. W. Landry, M. Reitman, E. J.Cragoe, Jr., and Q. Al-Awqati. J. Gen. Physiol. 90:779, (1987).

23. L. Virag, B. Mets, S. Jamdar, J. of Chromatography B. 681 263(1996).

24. A quantitative estimate of the conversion of cocaine to ecgoninemethyl ester by Mab 15A10 cannot be made directly from single in vivomeasurements of plasma concentrations due to differences in the kineticsof distribution and elimination for cocaine and ecgonine methyl ester:M. J. Chow, J. J. Ambre, T. I. Ruo, A. J. Atkinson, Jr., D. J. Bowsherand M. W. Fischman. Clin. Pharmacol. Ther. 38:318 (1985); J. Ambre, J.Nelson, S. Belknap, T. I. Rho. J. Anal. Toxicol. 12:301 (1988).

25. J. D. Stewart, V. A. Roberts, N. R. Thomas, E. D. Getzoff, S. J.Benkovic, J. Biochem. 33, 1994 (1994); E. Baldwin, P. G. Schultz.Science 245, 1104 (1989); C. J. Benkovic, J. Annu. Rev. Biochem. 61, 29(1992); D. Y. Jackson, J. R. Prudent, E. P. Baldwin, P. G. Schultz,Proc. Natl. Acad. Sci. 88, 58 (1991).

26. I. Benhar, E. A. Padlaw, S. H. Jung, B. Lee, I. Pastun, Proc. Natl.Acad. Sci. 91, 12051 (1994).

27. R. S. Hoffman, R. Morasco, L. R. Goldfrank, Clinical Toxicology 34,259 (1996).

28. M. Rocio, A. Cerrera, J. A. Ashley, L. H. Parsons, P. Wirsching, G.F. Koob, K. D. Janda, Nature 378, 727 (1995); B. S. Fox, K. M. Kantak,M. A. Edwards, K. M. Black, B. K. Bollinger, A. J. Botka, T. L. French,T. L. Thompson, V. C. Schad, J U. L. Greenstein, M. L. Gefter, M. A.Exley, P. A. Swain, T. J. Briner, Nature Medicine 2. 1129 (1996).

121 1 109 PRT Murinae gen. sp. 1 Ala Val Val Thr Gln Glu Ser Ala Leu ThrThr Trp Pro Gly Glu Thr 1 5 10 15 Val Thr Leu Thr Cys Arg Ser Ser ThrGly Thr Ile Thr Thr Ser Asn 20 25 30 Tyr Ala Asn Trp Val Gln Glu Lys ProAsp His Leu Phe Ser Gly Leu 35 40 45 Ile Gly Ile Asn Asn Asn Arg Pro ProGly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Leu Ile Gly Asp Lys Ala ValLeu Thr Ile Thr Gly Ala Gln 65 70 75 80 Thr Glu Asp Glu Ala Ile Tyr PheCys Ala Leu Trp Tyr Ser Asn His 85 90 95 Trp Val Phe Gly Gly Gly Thr LysLeu Thr Val Leu Gly 100 105 2 109 PRT Murinae gen.sp. 2 Ala Val Val ThrGln Glu Ser Ala Leu Thr Thr Arg Pro Gly Glu Thr 1 5 10 15 Val Thr LeuThr Cys Arg Ser Ser Ala Gly Thr Ile Thr Thr Ser Asn 20 25 30 Tyr Ala AsnTrp Val Gln Glu Lys Pro Asp His Leu Phe Ser Gly Leu 35 40 45 Ile Gly ValAsn Asn Asn Arg Pro Pro Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser LeuIle Gly Asp Thr Ala Ala Leu Thr Ile Thr Gly Ala Gln 65 70 75 80 Thr GluAsp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn His 85 90 95 Trp ValPhe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 3 109 PRT Murinaegen. Sp. 3 Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly GluThr 1 5 10 15 Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Thr Ile Thr SerAsp Asn 20 25 30 Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe SerGly Leu 35 40 45 Ile Gly Val Asn Asn Tyr Arg Pro Pro Gly Val Pro Ala ArgPhe Ser 50 55 60 Gly Ser Leu Thr Gly Asp Lys Ala Val Leu Thr Ile Thr GlyAla Gln 65 70 75 80 Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp TyrSer Asn His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly100 105 4 98 PRT Murinae gen.sp. 4 Thr Arg Ala Gly Glu Thr Val Thr ThrCys Arg Ser Ser Ser Gly Thr 1 5 10 15 Ile Thr Ala Asn Asn Tyr Gly SerTrp Val Gln Glu Lys Pro Asp His 20 25 30 Leu Phe Thr Gly Leu Ile Gly ValSer Asn Asn Arg Gly Pro Gly Val 35 40 45 Pro Ala Arg Phe Ser Gly Ser LeuIle Gly Asp Lys Ala Val Leu Thr 50 55 60 Ile Thr Gly Gly Gln Thr Glu AspGlu Ala Ile Tyr Phe Cys Ala Leu 65 70 75 80 Trp Asn Ser Asn His Phe ValPhe Gly Gly Gly Thr Lys Leu Thr Val 85 90 95 Leu Gly 5 113 PRT Murinaegen. Sp. 5 Asp Ile Val Met Thr Gln Asp Glu Leu Ser Asn Pro Val Thr SerGly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu LeuTyr Arg 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro GlyArg Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Met Ser Thr Arg Ser Ser GlyVal Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuGlu Ile 65 70 75 80 Ser Arg Val Lys Ala Glu Asp Val Gly Val Tyr Tyr CysGln His Phe 85 90 95 Val Asp Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys LeuGlu Ile Lys 100 105 110 Arg 6 113 PRT Murinae gen. sp. 6 Asp Met Val MetThr Gln Asp Glu Leu Ser Asn Pro Val Thr Ser Gly 1 5 10 15 Glu Ser ValSer Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu Tyr Arg 20 25 30 Asp Gly LysThr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Arg Ser 35 40 45 Pro Gln LeuLeu Ile Tyr Leu Met Ser Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile 65 70 75 80 Ser ArgVal Lys Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Phe 85 90 95 Glu AspTyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg7 113 PRT Murinae gen. sp 7 Asp Met Val Met Thr Gln Asp Glu Leu Ser AsnPro Val Thr Ser Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser SerArg Ser Leu Leu Tyr Arg 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe LeuGln Arg Pro Gly Arg Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Met Ser ThrArg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Glu Ile 65 70 75 80 Ser Arg Val Lys Ala Glu Asp Val GlyVal Tyr Tyr Cys Gln His Phe 85 90 95 Val Asp Tyr Pro Phe Thr Phe Gly SerGly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 8 113 PRT Murinae gen. sp. 8Asp Ile Val Ile Thr Gln Asp Glu Leu Ser Asn Pro Val Thr Ser Gly 1 5 1015 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Glu 20 2530 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 4045 Pro His Leu Leu Ile Tyr Leu Met Ser Thr Arg Ala Ser Gly Val Ser 50 5560 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile 65 7075 80 Ser Arg Val Lys Ala Glu Asp Val Gly Ala Tyr Tyr Cys Gln Gln Phe 8590 95 Val Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Arg100 105 110 Arg 9 114 PRT Murinae gen. sp. 9 Glu Leu Val Met Thr Gln SerPro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile SerCys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asp Gly Lys Thr Tyr LeuAsn Trp Phe Phe Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile TyrLeu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly SerGly Ser Gly Lys Asp Phe Thr Leu Lys Glu 65 70 75 80 Ile Ser Arg Val GluAla Glu Asp Leu Gly Leu Tyr Tyr Cys Val Gln 85 90 95 Gly Tyr Thr Phe ProLeu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys Arg 10 117PRT Murinae gen. sp. 10 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu ValLys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly AsnSer Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Thr Trp Ile Arg Gln Phe Pro GlyAsn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg His Ile Tyr Gly Thr ArgTyr Asn Pro Ser Leu 50 55 60 Ile Ser Arg Ile Ser Ile Thr Arg Asp Thr SerLys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asp Ser Val Thr Ala Glu AspThr Ala Thr Tyr Tyr Cys 85 90 95 Val Arg Tyr His Tyr Tyr Gly Ser Ala TyrTrp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 11 117 PRTMurinae gen. Sp. 11 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val LysPro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Asn SerIle Thr Ser Asp 20 25 30 Tyr Ala Trp Thr Trp Ile Arg Gln Phe Pro Gly AsnLys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg His Ile Tyr Gly Thr Arg TyrAsn Pro Ser Leu 50 55 60 Ile Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser LysAsn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asp Ser Val Thr Ala Glu Asp ThrAla Thr Tyr Tyr Cys 85 90 95 Val Arg Tyr His Tyr Tyr Gly Ser Ala Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 12 117 PRTMurinae gen.sp. 12 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val LysPro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Asn SerIle Thr Ser Asp 20 25 30 Tyr Ala Trp Thr Trp Ile Arg Lys Phe Pro Gly AsnLys Leu Glu Trp 35 40 45 Leu Gly Tyr Ile Arg His Ile Tyr Gly Thr Arg TyrAsn Pro Ser Leu 50 55 60 Ile Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser LysAsn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asp Ser Val Thr Ala Glu Asp ThrAla Thr Tyr Tyr Cys 85 90 95 Val Arg Tyr His Tyr Tyr Gly Ser Ala Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 13 117 PRTMurinae gen. sp. 13 Asp Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val LysPro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr SerIle Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly AsnArg Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg Tyr Ser Gly Ile Thr Arg TyrAsn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser LysAsn Lys Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp ThrAla Thr Tyr Tyr Cys 85 90 95 Val Arg Ile His Tyr Tyr Gly Tyr Gly Asn TrpGly Gln Gly Thr Thr 100 105 110 Leu Thr Gly Leu Pro 115 14 116 PRTMurinae gen. sp. 14 Asp Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val LysPro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr ProPhe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys SerLeu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Ser Asn Gly Gly Ile Phe TyrAsn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser SerAsn Thr Ala Phe 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp SerAla Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Gly Leu Phe Ala Tyr Trp GlyGln Gly Thr Leu Val 100 105 110 Thr Val Ser Glu 115 15 116 PRT Murinaegen. sp. 15 Glu Ile His Leu Gln Glu Ser Gly Glu Leu Val Lys Pro Gly AlaSer 1 5 10 15 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr SerAsp Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu GluTrp Ile 35 40 45 Gly Tyr Ile Asp Pro His Asn Gly Gly Ile Phe Tyr Asn GlnLys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Asn ThrAla Phe 65 70 75 80 Met His Leu Asn Val Leu Thr Ser Glu Asp Ser Ala ValTyr Tyr Cys 85 90 95 Ala Arg Gly Gly Gly Leu Phe Ala Tyr Trp Gly Arg GlyThr Leu Val 100 105 110 Thr Val Ser Ala 115 16 115 PRT Murinae gen. sp.16 Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 510 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Asp Tyr Asn 2025 30 Met Tyr Trp Val Lys Gln Asn His Gly Glu Ser Leu Glu Trp Ile Ala 3540 45 Tyr Ile Asp Pro Ser Asn Gly Asp Thr Arg Tyr Asn Gln Lys Phe Gln 5055 60 Gly Lys Ala Thr Val Thr Leu Asp Lys Ser Ser Ser Thr Ala Phe Met 6570 75 80 His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala85 90 95 Arg Gly Gly Gly Leu Phe Ala Phe Trp Gly Gln Gly Thr Leu Val Thr100 105 110 Val Ser Ala 115 17 116 PRT Murinae gen. sp. 17 Val Gln LeuLeu Glu Ser Gly Ala Glu Leu Val Met Pro Gly Ala Ser 1 5 10 15 Val LysMet Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His Trp 20 25 30 Met HisTrp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45 Thr IleAsp Leu Ser Asp Thr Tyr Thr Gly Tyr Asn Gln Asn Phe Lys 50 55 60 Gly ArgAla Thr Leu Thr Leu Asp Glu Ser Ser Asn Thr Ala Tyr Met 65 70 75 80 GlnLeu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ser 85 90 95 ArgArg Gly Tyr Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr Val Ser Ser 115 18 115 PRT Murinae gen. sp. 18 Val Gln Leu Leu GluSer Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Glu Leu SerCys Arg Thr Ser Gly Tyr Thr Phe Thr Thr Tyr Tyr 20 25 30 Ile Tyr Trp ValLys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45 Gly Met Asn ProGly Asn Gly Val Thr Tyr Phe Asn Glu Lys Phe Lys 50 55 60 Asn Arg Ala ThrLeu Thr Val Asp Arg Ser Ser Ser Ile Ala Tyr Met 65 70 75 80 Gln Leu SerSer Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr 85 90 95 Arg Val GlyAsn Leu Phe Ala Tyr Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val SerAla 115 19 16 PRT Murinae gen. sp. 19 Arg Ser Ser Arg Ser Leu Leu TyrArg Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 20 7 PRT Murinae gen. sp. 20Leu Met Ser Thr Arg Ser Ser 1 5 21 9 PRT Murinae gen.sp. 21 Gln His PheVal Asp Tyr Pro Phe Thr 1 5 22 16 PRT Murinae gen. sp. 22 Arg Ser SerLys Ser Leu Leu Tyr Glu Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 23 7 PRTMurinae gen. sp. 23 Leu Met Ser Thr Arg Ala Ser 1 5 24 9 PRT Murinaegen. sp. 24 Gln His Phe Glu Asp Tyr Pro Phe Thr 1 5 25 16 PRT Murinaegen. sp. 25 Arg Ser Ser Lys Ser Leu Leu Tyr Glu Asp Gly Lys Thr Tyr LeuAsn 1 5 10 15 26 7 PRT Murinae gen. sp. 26 Leu Met Ser Thr Arg Ala Ser 15 27 9 PRT Murinae gen. sp. 27 Gln Gln Phe Val Glu Tyr Pro Phe Thr 1 528 16 PRT Murinae gen. sp. 28 Arg Ser Ser Arg Ser Leu Leu Tyr Arg AspGly Lys Thr Tyr Leu Asn 1 5 10 15 29 7 PRT Murinae gen. sp. 29 Leu MetSer Thr Arg Ala Ser 1 5 30 9 PRT Murinae gen. sp. 30 Gln His Phe Glu AspTyr Pro Phe Thr 1 5 31 14 PRT Murinae gen. sp. 31 Arg Ser Ser Thr GlyThr Ile Thr Thr Ser Asn Tyr Ala Asn 1 5 10 32 7 PRT Murinae gen. sp. 32Ile Asn Asn Asn Arg Pro Pro 1 5 33 9 PRT Murinae gen. sp. 33 Ala Leu TrpTyr Ser Asn His Trp Val 1 5 34 14 PRT Murinae gen. sp. 34 Arg Ser SerAla Gly Thr Ile Thr Thr Ser Asn Tyr Ala Asn 1 5 10 35 7 PRT Murinae gen.sp. 35 Val Asn Asn Asn Arg Pro Pro 1 5 36 9 PRT Murinae gen. sp. 36 AlaLeu Trp Tyr Ser Asn His Trp Val 1 5 37 14 PRT Murinae gen. sp. 37 ArgSer Ser Thr Gly Thr Ile Thr Ser Asp Asn Tyr Ala Asn 1 5 10 38 7 PRTMurinae gen. sp. 38 Val Asn Asn Tyr Arg Pro Pro 1 5 39 9 PRT Murinaegen. sp. 39 Ala Leu Trp Tyr Ser Asn His Trp Val 1 5 40 14 PRT Murinaegen. sp. 40 Arg Ser Ser Ser Gly Thr Ile Thr Ala Asn Asn Tyr Gly Ser 1 510 41 7 PRT Murinae gen. sp. 41 Val Ser Asn Asn Arg Gly Pro 1 5 42 9 PRTMurinae gen. sp. 42 Ala Leu Trp Asn Ser Asn His Phe Val 1 5 43 16 PRTMurinae gen. sp. 43 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Asp Gly Lys ThrTyr Leu Asn 1 5 10 15 44 7 PRT Murinae gen. sp. 44 Leu Val Ser Lys LeuAsp Ser 1 5 45 9 PRT Murinae gen. sp. 45 Val Gln Gly Tyr Thr Phe Pro LeuThr 1 5 46 6 PRT Murinae gen. sp. 46 Ser Asp Tyr Ala Trp Thr 1 5 47 16PRT Murinae gen. sp. 47 Tyr Ile Arg His Ile Tyr Gly Thr Arg Tyr Asn ProSer Leu Ile Ser 1 5 10 15 48 8 PRT Murinae gen. sp. 48 Tyr His Tyr TyrGly Ser Ala Tyr 1 5 49 6 PRT Murinae gen. sp. 49 Ser Asp Tyr Ala Trp Thr1 5 50 16 PRT Murinae gen. sp. 50 Tyr Ile Arg His Ile Tyr Gly Thr ArgTyr Asn Pro Ser Leu Ile Ser 1 5 10 15 51 8 PRT Murinae gen. sp. 51 TyrHis Tyr Tyr Gly Ser Ala Tyr 1 5 52 6 PRT Murinae gen. sp. 52 Ser Asp TyrAla Trp Asn 1 5 53 16 PRT Murinae gen. sp. 53 Tyr Ile Arg Tyr Ser GlyIle Thr Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 54 8 PRT Murinae gen.sp. 54 Ile His Tyr Tyr Gly Tyr Gly Asn 1 5 55 6 PRT Murinae gen. sp. 55Ser Asp Tyr Ala Trp Thr 1 5 56 16 PRT Murinae gen. sp. 56 Tyr Ile ArgHis Ile Tyr Gly Thr Arg Tyr Asn Pro Ser Leu Ile Ser 1 5 10 15 57 8 PRTMurinae gen. sp. 57 Tyr His Tyr Tyr Gly Ser Ala Tyr 1 5 58 5 PRT Murinaegen. sp. 58 Asp Tyr Asn Met Tyr 1 5 59 17 PRT Murinae gen. sp. 59 TyrIle Asp Pro Ser Asn Gly Gly Ile Phe Tyr Asn Gln Lys Phe Lys 1 5 10 15Gly 60 7 PRT Murinae gen. sp. 60 Gly Gly Gly Leu Phe Ala Tyr 1 5 61 5PRT Murinae gen. sp.; 61 Asp Tyr Asn Met Tyr 1 5 62 17 PRT Murinae gen.sp. 62 Tyr Ile Asp Pro His Asn Gly Gly Ile Phe Tyr Asn Gln Lys Phe Lys 15 10 15 Gly 63 7 PRT Murinae gen. sp. 63 Gly Gly Gly Leu Phe Ala Tyr 1 564 5 PRT Murinae gen. sp. 64 Asp Tyr Asn Met Tyr 1 5 65 17 PRT Murinaegen. sp. 65 Tyr Ile Asp Pro Ser Asn Gly Asp Thr Phe Tyr Asn Gln Lys PheGln 1 5 10 15 Gly 66 7 PRT Murinae gen. sp. 66 Gly Gly Gly Leu Phe AlaPhe 1 5 67 5 PRT Murinae gen. sp. 67 Thr Tyr Tyr Ile Tyr 1 5 68 17 PRTMurinae gen. sp. 68 Gly Met Asn Pro Gly Asn Gly Val Thr Tyr Phe Asn GluLys Phe Lys 1 5 10 15 Asn 69 7 PRT Murinae gen. sp. 69 Val Gly Asn LeuPhe Ala Tyr 1 5 70 5 PRT Murinae gen. sp. 70 Asp His Trp Met His 1 5 7117 PRT Murinae gen. sp. 71 Thr Ile Asp Leu Ser Asp Thr Tyr Thr Gly TyrAsn Gln Asn Phe Lys 1 5 10 15 Gly 72 5 PRT Murinae gen. sp. 72 Arg GlyPhe Asp Tyr 1 5 73 14 PRT Murinae gen.sp. CHAIN (4)..(10) X at positions4, 9, 10 represents any amino acid 73 Arg Ser Ser Xaa Gly Thr Ile ThrXaa Xaa Asn Tyr Ala Asn 1 5 10 74 7 PRT Murinae gen. sp. CHAIN (1)..(1)X at position 1 represent any amino acid 74 Xaa Asn Asn Tyr Arg Pro Pro1 5 75 9 PRT Murinae gen. sp. 75 Ala Leu Trp Tyr Ser Asn His Trp Val 1 576 5 PRT Murinae gen. sp. 76 Asp Tyr Asn Met Tyr 1 5 77 17 PRT Murinaegen. sp. CHAIN (5)..(16) X at positions 5, 8,9,16 represents any aminoacid 77 Tyr Ile Asp Pro Xaa Asn Gly Xaa Xaa Phe Tyr Asn Gln Lys Phe Xaa1 5 10 15 Gly 78 7 PRT Murinae gen. sp. CHAIN (7)..(7) X at position 7represents any amino acid 78 Gly Gly Gly Leu Phe Ala Xaa 1 5 79 16 PRTMurinae gen. sp. CHAIN (4)..(9) X at positions 4 and 9 represents anyamino acid 79 Arg Ser Ser Xaa Ser Leu Leu Tyr Xaa Asp Gly Lys Thr TyrLeu Asn 1 5 10 15 80 7 PRT Murinae gen.sp. CHAIN (6)..(6) X at position6 represents any amino acid 80 Leu Met Ser Thr Arg Xaa Ser 1 5 81 9 PRTMurinae gen. sp. CHAIN (2)..(5) X at positions 2, 4 and 5 represents anyamino acid 81 Gln Xaa Phe Xaa Xaa Tyr Pro Phe Thr 1 5 82 6 PRT Murinaegen. sp. CHAIN (6)..(6) X at position 6 represents any amino acid 82 SerAsp Tyr Ala Trp Xaa 1 5 83 16 PRT Murinae gen. sp. CHAIN (4)..(15) X atposition 4,5, 6, 7, and 15 represents any amino acid 83 Tyr Ile Arg XaaXaa Xaa Xaa Thr Arg Tyr Asn Pro Ser Leu Xaa Ser 1 5 10 15 84 8 PRTMurinae gen. sp. CHAIN (1)..(8) X at positions 1, 6, 7, and 8 representsany amino acid 84 Xaa His Tyr Tyr Gly Xaa Xaa Xaa 1 5 85 330 DNA Murine85 tctggacctg agctggtgaa gcctggggct tcagtgaagg tatcctgtaa ggcttctggt 60tattcattca ctgactacaa tatgtactgg gtgaagcaga accatggaga gagccttgaa 120tggattgcat atattgatcc ttccaatggt gatactttct acaaccagaa attccagggc 180aaggccacag tgactcttga caagtcctcc agtacagcct tcatgcatct caacagcctg 240acatctgagg actctgcagt ctattactgt gcaagagggg ggggcctgtt tgctttctgg 300gggcaaggga ctctggtcac tgtctctgca 330 86 110 PRT Murine 86 Ser Gly ProGlu Leu Val Lys Pro Gly Ala Ser Val Lys Val Ser Cys 1 5 10 15 Lys AlaSer Gly Tyr Ser Phe Thr Asp Tyr Asn Met Tyr Trp Val Lys 20 25 30 Gln AsnHis Gly Glu Ser Leu Glu Trp Ile Ala Tyr Ile Asp Pro Ser 35 40 45 Asn GlyAsp Thr Phe Tyr Asn Gln Lys Phe Gln Gly Lys Ala Thr Val 50 55 60 Thr LeuAsp Lys Ser Ser Ser Thr Ala Phe Met His Leu Asn Ser Leu 65 70 75 80 ThrSer Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Gly Leu 85 90 95 PheAla Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 87 360DNA Murine; V_segment (16)..(356) n at any position represents anynucleotide including c,g,t,a,u 87 gtcgcatgct cccggncgnc atggncgcgggattgggaat tccacgaggc cgggggagac 60 agtcacactc acttgtcgtt caagtgctgggactattaca actagtaact atgccaactg 120 ggtccaagaa aaaccagatc atttattcagtggtctaata ggtgttaaca acaaccgacc 180 tccaggtgtt cctgccagat tctcaggctccctgattgga gacacggctg ccctcaccat 240 cacaggggca cagactgagg atgaggcaatatatttctgt gctctatggt acagcaacca 300 ctgggtgttc ggtggaggaa ccaaactgactgtcctaggc cagcccaagt cttcgncatc 360 88 99 PRT Murine 88 Thr Arg Pro GlyGlu Thr Val Thr Leu Thr Cys Arg Ser Ser Ala Gly 1 5 10 15 Thr Ile ThrThr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp 20 25 30 His Leu PheSer Gly Leu Ile Gly Val Asn Asn Asn Arg Pro Pro Gly 35 40 45 Val Pro AlaArg Phe Ser Gly Ser Leu Ile Gly Asp Thr Ala Ala Leu 50 55 60 Thr Ile ThrGly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala 65 70 75 80 Leu TrpTyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 85 90 95 Val LeuGly 89 419 DNA Murine 89 gaattcggca cgagcaggaa ctacaggtgt cactctgagatccacctgca gcagtctgga 60 cctgagctgg tgaagcctgg ggcttcagtg aagttatcctgcaaggcttc tggttactca 120 ttcactgact acaacatgta ctgggtgaaa cagagccatggaaagagcct tgagtggatt 180 ggatatattg atcctcacaa tggtggtatt ttctacaaccagaagttcaa gggcagggcc 240 acattgactg ttgacaagtc ctccaacaca gccttcatgcatctcaacag cctgacatct 300 gaggactctg cagtctatta ctgtgcaaga ggggggggcctgtttgctta ctggggccga 360 gggactctgg tcactgtctc tgcagccaaa acgacacccccatctgtcta tccactggc 419 90 116 PRT Murine 90 Glu Ile His Leu Gln GlnSer Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu SerCys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Tyr Trp ValLys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp ProHis Asn Gly Gly Ile Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala ThrLeu Thr Val Asp Lys Ser Ser Asn Thr Ala Phe 65 70 75 80 Met His Leu AsnSer Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly GlyGly Leu Phe Ala Tyr Trp Gly Arg Gly Thr Leu Val 100 105 110 Thr Val SerAla 115 91 360 DNA Murine V_segment (16)..(356) n at any positionrepresents any nucleotide including c,g,t,a,u 91 gtcgcatgct cccggncgccatggncgcgg gattgggaat tccacgtggc cgggggagac 60 agtcacactc acttgtcgctcaagtactgg gactattaca actagtaact atgccaactg 120 ggtccaagaa aaaccagatcatttattcag tggtctgata ggtattaaca acaaccgacc 180 tccaggtgtt cctgccagattctcaggctc cctgattgga gacaaggctg tcctcaccat 240 cacaggggca cagactgaggatgaggcaat atatttctgt gctctatggt acagcaacca 300 ctgggtgttc ggtggaggaaccaaactgac tgtcctaggc cagcccaagt cttcgncatc 360 92 99 PRT Murine 92 ThrTrp Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly 1 5 10 15Thr Ile Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp 20 25 30His Leu Phe Ser Gly Leu Ile Gly Ile Asn Asn Asn Arg Pro Pro Gly 35 40 45Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Val Leu 50 55 60Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala 65 70 7580 Leu Trp Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 85 9095 Val Leu Gly 93 360 DNA Murine 93 ggtccagctg ctcgagtctg gacctgagctggtgaagcct ggggcttcag tgaagttatc 60 ctgcaaggct tctggttacc cattcactgactacaacatg tactgggtga agcagagcca 120 tggaaagagc cttgagtgga ttggatatattgatccttcc aatggtggta ttttttacaa 180 ccagaagttc aagggcaggg ccacattgactgttgacaag tcctccaaca cagccttcat 240 gcatctcaac agcctgacat ctgaggactctgcagtctat tactgtgcaa gagggggggg 300 cctgtttgct tactggggcc aagggactctggtcactgtc tctgaagcca aaacgaaacc 360 94 110 PRT Murine 94 Ser Gly ProGlu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys 1 5 10 15 Lys AlaSer Gly Tyr Pro Phe Thr Asp Tyr Asn Met Tyr Trp Val Lys 20 25 30 Gln SerHis Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Asp Pro Ser 35 40 45 Asn GlyGly Ile Phe Tyr Asn Gln Lys Phe Lys Gly Arg Ala Thr Leu 50 55 60 Thr ValAsp Lys Ser Ser Asn Thr Ala Phe Met His Leu Asn Ser Leu 65 70 75 80 ThrSer Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Gly Leu 85 90 95 PheAla Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Glu 100 105 110 95 360DNA Murine 95 aggcggccgc actagtgatt gggaattcca cgagggcggg ggagacagtcacactcactt 60 gtcgctcaag tagtgggact attacagcta ataactatgg cagctgggtccaggaaaagc 120 cagatcattt attcactggt ctaataggtg ttagcaacaa ccgaggtccaggtgttcctg 180 ccagattctc aggctcccta attggagaca aggctgtcct caccatcacgggggggcaga 240 ctgaggatga ggcaatttat ttctgtgctc tatggaacag caaccatttcgtgttcggtg 300 gaggaaccaa actgactgtc ctagggcaga ccaagtcttt cggcatcaagcaccctgttt 360 96 100 PRT Murine 96 Thr Arg Ala Gly Glu Thr Val Thr LeuThr Cys Arg Ser Ser Ser Gly 1 5 10 15 Thr Ile Thr Ala Asn Asn Tyr GlySer Trp Val Gln Glu Lys Pro Asp 20 25 30 His Leu Phe Thr Gly Leu Ile GlyVal Ser Asn Asn Arg Gly Pro Gly 35 40 45 Val Pro Ala Arg Phe Ser Gly SerLeu Ile Gly Asp Lys Ala Val Leu 50 55 60 Thr Ile Thr Gly Gly Gln Thr GluAsp Glu Ala Ile Tyr Phe Cys Ala 65 70 75 80 Leu Trp Asn Ser Asn His PheVal Phe Gly Gly Gly Thr Lys Leu Thr 85 90 95 Val Leu Gly Gln 100 97 419DNA Murine 97 ccattgggcc cgacgtcgca tgctcccggc cgccatggcc gcgggattaggtccaacttc 60 tcgagtctgg ggctgaactg gtgaagcctg gggcttcagt ggagttgtcctgcaggactt 120 ctggctacac cttcaccacc tactatattt actgggtaaa acagaggcctggacaaggcc 180 ttgagtggat tggggggatg aatcctggca atggtgttac ttacttcaatgaaaaattca 240 agaacagggc cacactgact gtggacagat cctccagcat tgcctacatgcaactcagca 300 gcctgacatc tgaggactct gcggtctatt actgtacacg ggtgggtaactctttgctta 360 ctggggccga gggactctgg tcactgtctc tgcagccaaa acgacaccccactttctat 419 98 115 PRT Murine 98 Val Gln Leu Leu Glu Ser Gly Ala GluLeu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Glu Leu Ser Cys Arg Thr SerGly Tyr Thr Phe Thr Thr Tyr Tyr 20 25 30 Ile Tyr Trp Val Lys Gln Arg ProGly Gln Gly Leu Glu Trp Ile Gly 35 40 45 Gly Met Asn Pro Gly Asn Gly ValThr Tyr Phe Asn Glu Lys Phe Lys 50 55 60 Asn Arg Ala Thr Leu Thr Val AspArg Ser Ser Ser Ile Ala Tyr Met 65 70 75 80 Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Tyr Cys Thr 85 90 95 Arg Val Gly Asn Ser Leu LeuThr Gly Ala Glu Gly Leu Trp Ser Leu 100 105 110 Ser Leu Gln 115 99 339DNA Murine 99 gatattgtga tgacccagga tgaactctcc aatcctgtca cttctggagaatcagtttcc 60 atctcctgca ggtctagtag gagtctccta tatagggatg ggaagacatacttgaattgg 120 tttctgcaga gaccaggacg atctcctcaa ctcctgatct atttgatgtccacccgttca 180 tcaggagtct cagaccggtt tagtggcagt gggtcaggaa cagatttcaccctggaaatc 240 agtagagtga aggctgagga tgtgggtgtg tattactgtc aacactttgtagactatcca 300 ttcacgttcg gctcggggac aaagttggag ataaaacgg 339 100 113PRT Murine 100 Asp Ile Val Met Thr Gln Asp Glu Leu Ser Asn Pro Val ThrSer Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Arg Ser LeuLeu Tyr Arg 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg ProGly Arg Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Met Ser Thr Arg Ser SerGly Val Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrLeu Glu Ile 65 70 75 80 Ser Arg Val Lys Ala Glu Asp Val Gly Val Tyr TyrCys Gln His Phe 85 90 95 Val Asp Tyr Pro Phe Thr Phe Gly Ser Gly Thr LysLeu Glu Ile Lys 100 105 110 Arg 101 366 DNA Murine 101 gatgtgcagcttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactgtcactggcaa ttcaatcacc agtgattatg cctggacctg gatccggcag 120 tttccaggaaacaaactgga gtggatgggc tacataaggc acatttatgg cactaggtac 180 aacccttctctcataagtcg aatctctatc actcgagaca cgtccaagaa ccagttcttc 240 ctgcagttggattctgtgac tgctgaggac acagccacat attattgtgt aagatatcat 300 tactacggttcggcttactg gggccaaggg actctggtca ctgtctctgc agccaaaacg 360 acaccc 366102 122 PRT Murine 102 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu ValLys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly AsnSer Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Thr Trp Ile Arg Gln Phe Pro GlyAsn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg His Ile Tyr Gly Thr ArgTyr Asn Pro Ser Leu 50 55 60 Ile Ser Arg Ile Ser Ile Thr Arg Asp Thr SerLys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asp Ser Val Thr Ala Glu AspThr Ala Thr Tyr Tyr Cys 85 90 95 Val Arg Tyr His Tyr Tyr Gly Ser Ala TyrTrp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala Ala Lys Thr ThrPro 115 120 103 368 DNA MURINE 103 gatatggtga tgacgcaaga tgaactctccaatcctgtca cttctggaga atcagtttcc 60 atctcctgca ggtctagtag gagtctcctatatagggatg ggaagacata cttgaattgg 120 tttctgcaga gaccaggacg atctcctcaactcctgatct atttgatgtc cacccgtgca 180 tcaggagtct cagaccggtt tagtggcagtgggtcaggaa cagatttcac cctggaaatc 240 agtagagtga aggctgagga tgtgggtgtgtattactttc aacactttga agactatcca 300 ttcacgttcg gctcggggac aaaattggagataaaacggg ctgatgctgc accaactgta 360 tccatctt 368 104 113 PRT Murine 104Asp Met Val Met Thr Gln Asp Glu Leu Ser Asn Pro Val Thr Ser Gly 1 5 1015 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu Tyr Arg 20 2530 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Arg Ser 35 4045 Pro Gln Leu Leu Ile Tyr Leu Met Ser Thr Arg Ala Ser Gly Val Ser 50 5560 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile 65 7075 80 Ser Arg Val Lys Ala Glu Asp Val Gly Val Tyr Tyr Phe Gln His Phe 8590 95 Glu Asp Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys100 105 110 Arg 105 366 DNA Murine 105 gacgtgcagt tgcaggagtc gggacctggcctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcaa ttcaatcaccagtgattatg cctggacctg gatccggcag 120 tttccaggaa acaaactgga gtggatgggctacataaggc acatttatgg cactaggtac 180 aacccttctc tcataagtcg aatctctatcactcgagaca cgtccaagaa ccagttcttc 240 ctgcagttgg attctgtgac tgctgaggacacagccacat attattgtgt aagatatcat 300 tactacggtt cggcttactg gggccaagggactctggtca ctgtctctgc agccaaaacg 360 acaccc 366 106 122 PRT Murine 106Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 1015 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Asn Ser Ile Thr Ser Asp 20 2530 Tyr Ala Trp Thr Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 4045 Met Gly Tyr Ile Arg His Ile Tyr Gly Thr Arg Tyr Asn Pro Ser Leu 50 5560 Ile Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 7075 80 Leu Gln Leu Asp Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 8590 95 Val Arg Tyr His Tyr Tyr Gly Ser Ala Tyr Trp Gly Gln Gly Thr Leu100 105 110 Val Thr Val Ser Ala Ala Lys Thr Thr Pro 115 120 107 368 DNAMurine 107 gatatggtga tgacgcaaga cgaactctcc aatcctgtca cttctggagaatcagtttcc 60 atctcctgca ggtctagtaa gagtctccta tatgaggatg ggaagacatacttgaattgg 120 tttctgcaga gaccaggaca atctcctcac ctcctgatct atttgatgtccacccgtgca 180 tcaggagtct cagaccggtt tagtggcagt gggtcaggaa cagatttcaccctggaaatc 240 agtagagtga aggctgagga tgtgggtgcg tattactgtc aacaatttgtagagtatcca 300 ttcacgttcg gctcggggac aaagttggaa ataagacggg ttgatgccgcaccaactgta 360 tccatctt 368 108 113 PRT Murine 108 Asp Met Val Met ThrGln Asp Glu Leu Ser Asn Pro Val Thr Ser Gly 1 5 10 15 Glu Ser Val SerIle Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Glu 20 25 30 Asp Gly Lys ThrTyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro His Leu LeuIle Tyr Leu Met Ser Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile 65 70 75 80 Ser Arg ValLys Ala Glu Asp Val Gly Ala Tyr Tyr Cys Gln Gln Phe 85 90 95 Val Glu TyrPro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Arg 100 105 110 Arg 109420 DNA Murine V_segment (21)..(56) n at any position represents anynucleotide including c,g,t,a,u 109 cattgggccc acgtcgaatg ntcccggncgncatggncgn gggattgana gggggncgga 60 gctggtgaag ccttctcagt ctctgtccctcacctgcact gtcactggct actcaatcac 120 cagtgattat gcctggaact ggatccggcagtttccagga aacagactgg agtggatggg 180 ctacataagg tacagtggta tcactaggtacaacccatct ctcaaaagtc gaatctctat 240 cactcgagac acatccaaga acaagttcttcctgcagtta aattctgtga ctactgagga 300 cacagccact tattactgtg taagaattcattactacggc tacggcaact gggggcaagg 360 caccactctc acaggtcttc ctcaagagtctgggaagaaa tcccacccat cttccccact 420 110 108 PRT Murine 110 Glu Leu ValLys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr 1 5 10 15 Gly TyrSer Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe 20 25 30 Pro GlyAsn Arg Leu Glu Trp Met Gly Tyr Ile Arg Tyr Ser Gly Ile 35 40 45 Thr ArgTyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp 50 55 60 Thr SerLys Asn Lys Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu 65 70 75 80 AspThr Ala Thr Tyr Tyr Cys Val Arg Ile His Tyr Tyr Gly Tyr Gly 85 90 95 AsnTrp Gly Gln Gly Thr Thr Leu Thr Gly Leu Pro 100 105 111 420 DNA MurineV_segment (1)..(403) n at any position represents any nucleotideincluding c.g,t,a,u 111 nccttgggcc ganggcgcat gctcccggcc gccatggccgcgggattaga gcgatatggt 60 gatgacgcag gatgaactct ccaatcctgt cacttctggagaatcagttt ccatctcctg 120 caggtctagt aggagtctcc tatataggga tgggaagacatacttgaatt ggtttctgca 180 gagaccagga cgatctcctc aactcctgat ctatttgatgtccacccgtg catcaggagt 240 ctcagaccgg tttagtggca gtgggtcagg aacagatttcaccctggaaa tcagtagagt 300 gaaggctgag gatgtgggtg tgtattactg tcaacactttgtagactatc cattcacgtt 360 cggctcgggg acaaagttgg agataaaacg ggttgatgctgnancaactg tatccatctt 420 112 113 PRT Murine 112 Asp Met Val Met Thr GlnAsp Glu Leu Ser Asn Pro Val Thr Ser Gly 1 5 10 15 Glu Ser Val Ser IleSer Cys Arg Ser Ser Arg Ser Leu Leu Tyr Arg 20 25 30 Asp Gly Lys Thr TyrLeu Asn Trp Phe Leu Gln Arg Pro Gly Arg Ser 35 40 45 Pro Gln Leu Leu IleTyr Leu Met Ser Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile 65 70 75 80 Ser Arg Val LysAla Glu Asp Val Gly Val Tyr Tyr Cys Gln His Phe 85 90 95 Val Asp Tyr ProPhe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg 113 419DNA Murine V_segment (381)..(381) n at any position represents anynucleotide including c,g,t,a,u 113 ctagtgattg ctctagagcg acgtgcagttgcaggagtcg ggacctggac tggtgaaacc 60 ttctcagtct ctgtccctca cctgcactgtcactggtaat tcaatcacca gtgattatgc 120 ctggacctgg atccggaagt ttccaggaaacaaactggag tggttgggct acataaggca 180 catttatggc actaggtaca acccttctctcataagtcga atctctatca ctcgagacac 240 gtccaagaac cagttcttcc tgcagttggattctgtgact gctgaggaca cagccacata 300 ttattgtgta agatatcatt actacgggtcggcttactgg gggcaaggga ctctggtcac 360 tgtctctgca ggcaaaacga naccccatctgtctatcact ggccccggaa cgccgccag 419 114 117 PRT Murine 114 Asp Val GlnLeu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser LeuSer Leu Thr Cys Thr Val Thr Gly Asn Ser Ile Thr Ser Asp 20 25 30 Tyr AlaTrp Thr Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Leu GlyTyr Ile Arg His Ile Tyr Gly Thr Arg Tyr Asn Pro Ser Leu 50 55 60 Ile SerArg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 LeuGln Leu Asp Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 ValArg Tyr His Tyr Tyr Gly Ser Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ala 115 115 420 DNA Murine V_segment (3)..(43) n at anyposition represents any nucleotide including c,g,t,a,u 115 ttnaaggcccngacgccgca tagctcncgg ccgccatggc cgngggattc cagttccgag 60 ctcgtgatgacacagtctcc actcactttg tcggtaacca ttggacaacc agcctctatc 120 tcttgcaagtcaagtcagag cctcttatat agtgatggaa aaacctattt gaattggttc 180 ttccagaggccaggccagtc tccaaagcgc ctaatctatc tggtgtctaa actggactct 240 ggagtccctgacaggttcac tggcagtgga tcaggaaaag attttacact gaaaatcagc 300 agagtggaggctgaggattt gggactttat tactgcgttc aagggtacac atttccgctc 360 acgttcggtgctgggaccaa gctggagctg aaacgggtga tgctgaccaa cttgtttcat 420 116 113 PRTMurine 116 Glu Leu Val Met Thr Gln Ser Pro Leu Thr Leu Ser Val Thr IleGly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu LeuTyr Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Phe Gln Arg Pro GlyGln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser GlyVal Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Lys Asp Phe Thr LeuLys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr CysVal Gln Gly 85 90 95 Tyr Thr Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys LeuGlu Leu Lys 100 105 110 Arg 117 420 DNA Murine V_segment (37)..(414) nat any position represents any nucleotide including c,g,t,a,u 117ttgggcccgg acgtcgcatg ctcccggccg ccatggncgn gggattaggt ccaacttctc 60gagtctgggg ctgagcttgt gatgcctggg gcttcagtga agatgtcctg caaggcttct 120ggctacacat tcactgacca ctggatgcac tgggtgaagc agaggcctgg acaaggcctt 180gagtggatcg gaacgattga tctttctgat acttatactg gctacaatca aaacttcaag 240ggcagggcca cattgactct cgacgaatcc tccaacacag cctacatgca gctcagcagc 300ctgacatctg aggactctgc ggtctattac tgttcaagaa ggggctttga ctactggggg 360caaggcacca ctctcacagt ctcctcaggc aaaacgacaa ccccatcttg tctntccact 420118 113 PRT Murine 118 Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val MetPro Gly Ala Ser 1 5 10 15 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr ThrPhe Thr Asp His Trp 20 25 30 Met His Trp Val Lys Gln Arg Pro Gly Gln GlyLeu Glu Trp Ile Gly 35 40 45 Thr Ile Asp Leu Ser Asp Thr Tyr Thr Gly TyrAsn Gln Asn Phe Lys 50 55 60 Gly Arg Ala Thr Leu Thr Leu Asp Glu Ser SerAsn Thr Ala Tyr Met 65 70 75 80 Gln Leu Ser Ser Leu Thr Ser Glu Asp SerAla Val Tyr Tyr Cys Ser 85 90 95 Arg Arg Gly Phe Asp Tyr Trp Gly Gln GlyThr Thr Leu Thr Val Ser 100 105 110 Ser 119 280 PRT Murine 119 Met GluVal Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Ser 1 5 10 15 GlnSer Leu Ser Leu Thr Cys Thr Val Thr Gly Asn Ser Ile Thr Ser 20 25 30 AspTyr Ala Trp Thr Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu 35 40 45 TrpMet Gly Tyr Ile Arg His Ile Tyr Gly Thr Arg Tyr Asn Pro Ser 50 55 60 LeuIle Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe 65 70 75 80Phe Leu Gln Leu Asp Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Val Arg Tyr His Tyr Tyr Gly Ser Ala Tyr Trp Gly Gln Gly Thr 100 105110 Leu Val Thr Val Ser Ala Gly Met Gln Ser Gly Gly Gly Gly Ser Gly 115120 125 Gly Gly Gly Ser Gly Gly Ala Met Asp Ile Val Met Thr Gln Asp Glu130 135 140 Leu Ser Asn Pro Val Thr Ser Gly Glu Ser Val Ser Ile Ser CysArg 145 150 155 160 Ser Ser Arg Ser Leu Leu Tyr Arg Asp Gly Lys Thr TyrLeu Asn Trp 165 170 175 Phe Leu Gln Arg Pro Gly Arg Pro Pro Gln Leu LeuIle Tyr Leu Met 180 185 190 Ser Thr Arg Ser Ser Gly Val Ser Asp Arg PheSer Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Glu Ile Ser ArgVal Lys Ala Glu Asp Val 210 215 220 Gly Val Tyr Tyr Cys Gln His Phe ValAsp Tyr Pro Phe Thr Phe Gly 225 230 235 240 Ser Gly Thr Lys Leu Glu IleLys Arg Ala Asp Gly Ala Pro Thr Val 245 250 255 Ser Ile Phe Phe Pro ProSer Leu Asp Tyr Lys Asp Asp Asp Asp Lys 260 265 270 Leu Glu His His HisHis His His 275 280 120 360 DNA Murine 120 gctgttgtta ctcaggagtctgctctaact acatcacctg gtgaaacagt cacactcact 60 tgtcgctcaa gtactgggactattacaagt gataactatg ccaactgggt ccaagaaaaa 120 ccagatcatt tattcagtggtctaataggt gttaataatt accgacctcc aggtgttcct 180 gccagattct caggctccctgactggagac aaggctgtcc tcaccatcac aggggcacag 240 actgaggatg aggcaatatatttctgtgct ctatggtaca gcaaccactg ggtgttcggt 300 ggaggaacca aactgactgtcctaggccag cccaagtctt cgccatcagt caccctgttt 360 121 109 PRT Murine 121Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu Thr 1 5 1015 Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Thr Ile Thr Ser Asp Asn 20 2530 Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Ser Gly Leu 35 4045 Ile Gly Val Asn Asn Tyr Arg Pro Pro Gly Val Pro Ala Arg Phe Ser 50 5560 Gly Ser Leu Thr Gly Asp Lys Ala Val Leu Thr Ile Thr Gly Ala Gln 65 7075 80 Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn His 8590 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105

What is claimed is:
 1. A polypeptide comprising a light chain domainwhich comprises a complementarity determining region 1 having the aminoacid sequence RSSXGTITXXNYAN (Seq ID No: 73), a complementaritydetermining region 2 having the amino acid sequence XNNYRPP (Seq ID No:74) and a complementarity determining region 3 having the amino acidsequence ALWYSNHWV (Seq ID No: 75), interposed between appropriateframework regions, and linked to said light chain domain a heavy chaindomain which comprises a complementarity determining region 1 having theamino acid sequence DYNMY (Seq ID No: 76), a complementarity determiningregion 2 having the amino acid sequence YIDPXNGXIFYNQKFXG (Seq ID No:77) and a complementarity determining region 3 having the amino acidsequence GGGLFAX (Seq ID No: 78) interposed between appropriateframework regions, said polypeptide having a conformation suitable fordegrading cocaine.
 2. The polypeptide of claim 1, wherein the amino acidsequence of the complementarity determining region 1 of the light chainis RSSTGTITSDNYAN (Seq ID No. 37), the amino acid sequence of thecomplementarity determining region 2 of the light chain is VNNYRPP (SeqID No. 38) and the amino acid sequence of the complementaritydetermining region 3 of the light chain is ALWYSNHWV (Seq ID No. 39) andthe corresponding amino acid sequence of the complementarity determiningregion 1 of the heavy chain is DYNMY (Seq ID No: 64), the amino acidsequence of complementarity determining region 2 of the heavy chain isYIDPSNGDTFYNQKFQG (Seq ID No: 65) and complementarity determining region3 of the heavy chain is GGGLFAF (Seq ID No: 66).
 3. The polypeptide ofclaim 2, wherein the light chain domain comprises the amino acidsequence as set forth in Seq ID No:3 and the heavy chain comprises theamino acid sequence as set forth in Seq ID No:
 16. 4. The polypeptide ofclaim 1, wherein the amino acid sequence of the complementaritydetermining region 1 of the light chain is RSSAGTITTSNYAN (Seq ID No.34), the amino acid sequence of the complementarity determining region 2of the light chain having amino acid sequence is VNNNRPP (Seq ID No. 35)and the amino acid sequence of the complementarity determining region 3of the light chain is ALWYSNHWV (Seq ID No. 36) and the correspondingamino acid sequence of the complementarity determining region 1 of theheavy chain is DYNMY (Seq ID No: 61), the amino acid sequence of thecomplementarity determining region 2 of the heavy chain isYIDPHNGGIFYNQKFKG (Seq ID No: 62) and the amino acid sequence of thecomplementarity determining region 3 of the heavy chain is GGGLFAY (SeqID No: 63).
 5. The polypeptide of claim 4, wherein the light chaincomprises the amino acid sequence as set forth in Seq ID No:2 and theheavy chain comprises the amino acid sequence as set forth in Seq ID No:15.
 6. The polypeptide of claim 1, wherein the amino acid sequence ofthe complementarity determining region 1 of the light chain isRSSTGTITTSNYAN (Seq ID No. 31), the amino acid sequence of thecomplementarity determining region 2 of the light chain is INNNRPP (SeqID No. 32) and the amino acid sequence of the complementaritydetermining region 3 of the light chain is ALWYSNHWV (Seq ID No. 33) andthe corresponding amino acid sequence of the complementarity determiningregion 1 of the heavy chain is DYNMY (Seq ID No: 58), the amino acidsequence of the complementarity determining region 2 isYIDPSNGGIFYNQKFKG (Seq ID No: 59) and the amino acid sequence of thecomplementarity determining region 3 is GGGLFAY (Seq ID No: 60).
 7. Thepolypeptide of claim 6, wherein the light chain comprises the amino acidsequence as set forth in Seq ID No:1 and the heavy chain comprises theamino acid sequence as set forth in Seq ID No:
 14. 8. A polypeptidecomprising a light chain domain which comprises a complementaritydetermining region 1 having the amino acid sequence RSSSGTITANNYGS (SeqID No: 40), a complementarity determining region 2 having the amino acidsequence VSNNRGP (Seq ID No: 41) and a complementarity determiningregion 3 having the amino acid sequence ALWNSNHFV (Seq ID No: 42),interposed between appropriate framework regions, and linked to saidlight chain domain a heavy chain domain which comprises acomplementarity determining region 1 having the amino acid sequenceTYYIY (Seq ID No: 67), a complementarity determining region 2 having theamino acid sequence GMNPGNGVTYFNEKFKN (Seq ID No: 68) and acomplementarity determining region 3 having the amino acid sequenceVGNLFAY (Seq ID No: 69) interposed between appropriate frameworkregions, said polypeptide having a conformation suitable for degradingcocaine.
 9. The polypeptide of claim 8, wherein the light chaincomprises the amino acid sequence as set forth in Seq ID No:4 and theheavy chain comprises the amino acid sequence as set forth in Seq ID No:18.
 10. A polypeptide comprising a light chain domain which comprises acomplementarity determining region 1 having the amino acid sequenceRSSXSLLYXDGKTYLN (Seq ID No: 79), a complementarity determining region 2having the amino acid sequence LMSTRXS (Seq ID No: 80) and acomplementarity determining region 3 having the amino acid sequenceQXFXXYPFT (Seq ID No: 81), interposed between appropriate humanframework regions, and linked to said light chain domain a heavy chaindomain which comprises a complementarity determining region 1 having theamino acid sequence SDYAWX (Seq ID No: 82), a complementaritydetermining region 2 having the amino acid sequence YIRXXXXTRYNPSLXS(Seq ID No: 83) and a complementarity determining region 3 having theamino acid sequence XHYYGXXX (Seq ID No: 84) interposed betweenappropriate human framework regions, said polypeptide having aconformation suitable for degrading cocaine.
 11. The polypeptide ofclaim 10, wherein the amino acid sequence of the complementaritydetermining region 1 of the light chain is RSSRSLLYRDGKTYLN (Seq ID No.19), the amino acid sequence of the complementarity determining region 2of the light chain is LMSTRSS (Seq ID No. 20) and the amino acidsequence of the complementarity determining region 3 of the light chainis QHFVDYPFT (Seq ID No. 21) and the corresponding amino acid sequenceof the complementarity determining region 1 of the heavy chain is SDYAWT(Seq ID No: 46), the amino acid sequence of the complementaritydetermining region 2 of the heavy chain is YIRHIYGTRYNPSLIS (Seq ID No:47) and the amino acid sequence of the complementarity determiningregion 3 of the heavy chain is YHYYGSAY (Seq ID No: 48).
 12. Thepolypeptide of claim 11, wherein the light chain comprises the aminoacid sequence as set forth in Seq ID No:5 and the heavy chain comprisesthe amino acid sequence as set forth in Seq ID No:
 10. 13. Thepolypeptide of claim 10, wherein the amino acid sequence of thecomplementarity determining region 1 of the light chain isRSSKSLLYEDGKTYLN (Seq ID No. 22), the amino acid sequence of thecomplementarity determining region 2 of the light chain is LMSTRAS (SeqID No. 23), the amino acid sequence of the complementarity determiningregion 3 of the light chain is QHFEDYPFT (Seq ID No. 24) and thecorresponding amino acid of the complementarity determining region 1 ofthe heavy chain is SDYAWT (Seq ID No: 46), the amino acid sequence ofthe complementarity determining region 2 of the heavy chain isYIRHIYGTRYNPSLIS (Seq ID No: 47) and the amino acid sequence of thecomplementarity determining region 3 of the heavy chain is YHYYGSAY (SeqID No: 48).
 14. The polypeptide of claim 13, wherein the light chaincomprises the amino acid sequence as set forth in Seq ID No:6 and theheavy chain comprises the amino acid sequence as set forth in Seq ID No:11.
 15. The polypeptide of claim 10, wherein the amino acid of thecomplementarity determining region 1 of the light chain isRSSKSLLYEDGKTYLN (Seq ID No. 25), the amino acid sequence of thecomplementarity determining region 2 of the light chain is LMSTRAS (SeqID No. 26), and the amino acid sequence of the complementaritydetermining region 3 of the light chain is QQFVEYPFT (Seq ID No. 27) andthe corresponding amino acid of the complementarity determining region 1of the heavy chain is SDYAWN (Seq ID No: 52), the amino acid sequence ofthe complementarity determining region 2 of the heavy chain isYIRYSGITRYNPSLKS (Seq ID No: 53) and the amino acid sequence of thecomplementarity determining region 3 of the heavy chain is IHYYGYGN (SeqID No: 54).
 16. The polypeptide of claim 15, wherein the light chaincomprises the amino acid sequence as set forth in Seq ID No:8 and theheavy chain comprises the amino acid sequence as set forth in Seq ID No:13.
 17. The polypeptide of claim 10, wherein the amino acid sequence ofthe complementarity determining region 1 of the light chain isRSSRSLLYRDGKTYLN (Seq ID No. 28), the amino acid sequence of thecomplementarity determining region 2 of the light chain is LMSTRAS (SeqID No. 29), the amino acid sequence of the complementarity determiningregion 3 of the light chain QHFEDYPFT (Seq ID No. 30) and thecorresponding amino acid sequence of the complementarity determiningregion 1 of the heavy chain is SDYAWT (Seq ID No: 55), the amino acidsequence of the complementarity determining region 2 of the heavy chainis YIRHIYGTRYNPSLIS (Seq ID No: 56) and the amino acid sequence of thecomplementarity determining region 3 of the heavy chain is YHYYGSAY (SeqID No: 57).
 18. The polypeptide of claim 17, wherein the light chaincomprises the amino acid sequence as set forth in Seq ID No:7 and theheavy chain comprises the amino acid sequence as set forth in Seq ID No:12.
 19. A polypeptide comprising a light chain domain which comprises acomplementarity determining region 1 having the amino acid sequenceKSSQSLLYSDGKTYLN (Seq ID No: 43), a complementarity determining region 2having the amino acid sequence LVSKLDS (Seq ID No: 44) and acomplementarity determining region 3 having the amino acid sequenceVQGYTFPLT (Seq ID No: 45), interposed between appropriate frameworkregions, and linked to said light chain domain a heavy chain domainwhich comprises a complementarity determining region 1 having the aminoacid sequence DHWMH (Seq ID No: 70), a complementarity determiningregion 2 having the amino acid sequence TIDLSDTYTGYNQNFKG (Seq ID No:71) and a complementarity determining region 3 having the amino acidsequence RGFDY (Seq ID No: 72) interposed between appropriate frameworkregions, said polypeptide having a conformation suitable for degradingcocaine.
 20. The polypeptide of claim 19, wherein the light chaincomprises the amino acid sequence as set forth in Seq ID No:9 and theheavy chain comprises the amino acid sequence as set forth in Seq ID No:17.
 21. The polypeptide of claim 1, wherein the appropriate frameworkregions of the light chain domain are human framework regions and theappropriate framework regions of the heavy chain domain are humanframework regions.
 22. The polypeptide of claim 8, wherein theappropriate framework regions of the light chain domain are humanframework regions and the appropriate framework regions of the heavychain domain are human framework regions.
 23. The polypeptide of claim19, wherein the appropriate framework regions of the light chain domainare human framework regions and the appropriate framework regions of theheavy chain domain are human framework regions.